BMB 515 Electronic Course Pack Sessions 11 - 20

Carbohydrate Metabolism
MITOCHONDRIA AND
THE PYRUVATE DEHYDROGENASE COMPLEX
Required Reading:
Wilkins: p. 65-73 Chapter 3. Sections on Mitochondrion: Structure and Function
through The Pyruvate Dehydrogenase Complex: The bridging step between
glycolysis and the TCA cycle
Coursepack: CLINICAL CASE, Pyruvate Dehydrogenase Complex
Suggested Reading:
Ferrier: Chapter 9, I. Cycle Overview through II. A. 5. Mechanism of arsenic poisoning
Objectives:
1. Describe the structural features of mitochondria and the significance of these
structures in controlling metabolic pathways.
2. Explain the purpose of the pyruvate dehydrogenase complex, and why it is a key
regulatory point of metabolism.
3. Identify where the pyruvate dehydrogenase complex is located in the cell.
4. Explain the overall reaction steps (as fits the oxidation state pattern of metabolism) of
the pyruvate dehydrogenase complex, and how certain vitamin deficiencies would
affect this complex.
5. Explain when the pyruvate dehydrogenase complex is activated or inhibited.
Measurable Outcomes: You should be able to ...
A. Describe the structural features of mitochondria. (Obj. 1)

B. Describe the functional significance of the two membranes and the compartments
of mitochondria. (Obj. 1)
C. Identify the aerobic and anaerobic fates of pyruvate. {Obj. 1, 2, and 3)
D. Identify the two main steps of the pyruvate dehydrogenase complex as
applications of the oxidation states basic principles. {Obj. 4)
E. Identify the 3 catalytic components of the pyruvate dehydrogenase complex and
each of their cofactors, and their vitamin precursors. {Obj. 4)
F. Explain the regulation of the pyruvate dehydrogenase complex in terms of both of
its regulatory components (the inhibitors and activators of the complex) and how
it is hormonally controlled. {Obj. 5)
G. Describe the clinical features of pyruvate dehydrogenase deficiency. {Obj. 4 and 5)
FATES OF PYR U VATE
(J. LaPres and C. Wilkins, MSU)
(- O 2 )
T o g e t m a x im u m A T P fro m g lu c o s e (38 to ta l),

need to m o v e th e p y ru v a te s g e n e ra te d fro m g ly c o ly s is in to th e
VIII. MITOCHONDRIA: Structure and Function
A. Size: ~ 1 |J (micron); about the size of a bacterium (i.e. E. Coli)
Mobile: moves on the cytoskeleton, and can divide.
Figure 3.1, p. 66: Basic structure of a mitochondrion

https://commons.wikimedia.orq/wiki/File:Animal mitochondrion diagram en.svq
B. Outer Membrane
1. Contains_______________
2. Permeability:
C. Intermembrane space (a.k.a. inner membrane space)

________________are pumped into this space.
D. Inner Membrane
1. Highly folded: forms__________.
2. Permeability:
3. Location o f__________________________
4. Primary purpose:
E. Matrix
1. Location of many metabolic pathways
2. Has high concentration o f________________
3. Contains 10+ copies of th e ________________________
F. Functional Roles of the mitochondria. (Figure 3.2, p. 67)

1. Uses:
2. Produces:
3. Functions include:
IX. THE PYR U V A TE D E H Y D R O G E N A S E C O M P LE X : T he b rid g in g s te p
b e tw e e n g ly c o ly s is a n d th e T C A c y c le ( W ilkins, pgs. 68-73)
A. W hat?
B. W hy?
C. W here?
D. How?
1. F ig u re 3.3, p. 69: Net reaction of the PDH com plex
2. M ultienzym e com plex (is huge): has 5 different enzym es--

a. 3 catalytic, each having a prosthetic group (i.e. a coenzym e)
b. 2 regulatory
3. T a b le 3.1, p. 69: The three PDH com plex catalytic enzym es and
the ir overall reactions
REC ALL vitam in sources of the co-factors involved in this com plex:
a. T hiam ine pyrophosphate (TPP): Thiam ine (vit. B 1)
b. Lipoam ide is not a vitam in; the body m akes it.
c. Flavin adenine dinucleotide (FAD): Riboflavin (vit. B 2 )
d. C oenzym e A: P antothenic acid (vit. B 5 )
e. N icotinam ide adenine dinucleotide (N AD +): Niacin (vit. B 3 )
4. W hy such a large com plex?
a.
b.
c.
5. A c lin ic a l n o te : The lipoam ide cofactor of E 2 is the site o f inhibition

by a rs e n ic .
a. In the 1800s, arsenic w as one of the few treatm ents for syphilis;
and becam e a popular ton ic of preventive m edicine .
b. C harles Darwin m ay have suffered from chronic arsenic poisoning
(depression and stom ach problem s)
c. Relevant knowledge for today: M ichigan, particularly the thum b
area , is one of several states in w hich ground w a te r can becom e
contam inated with high levels of arsenic. People w ith w ell-w ater
supplies are recom m ended to check frequently fo r arsenic levels.
E. F ig u re 3.4, p. 70: The PDH com plex reaction as tw o main steps (as
applies to the O xidation States Flow Chart).
W HEN?
F. The tw o regulatory enzym es of the PDH com plex
1. K inase: phosphorylates t h e _____________________, which
the PDH com plex. The kinase is activated b y ___________
w hich ultim ately is a ______________effect on the com plex.
2. P hosphatase: C leaves off t h e _______________ , w h ic h ____

the PDH com plex. The phosphatase is A C T IV A TE D b y __
G. PDH com plex is also in h ib ite d by:

PDH com plex is also a c tiv a te d by:
H. This bridging step is a ke y re g u la to ry p o in t:

1. The conversion of pyruvate to acetyl CoA is a k e y ______
step in animals. T hus anim als are c a n n o t convert
2. C onversion o f the glucose carbons to acetyl C oA com m its them to

one of tw o paths (mainly):
3. Thus, PDH com plex activity controls the fate of pyruvate:

REQUIRED READING
CLINICAL CASE: PYRUVATE DEHYDROGENASE COMPLEX
A Case Study
Genetic defect in the pyruvate dehydrogenase complex. A full-term male infant failed to gain
weight, had episodes of vomiting, and showed metabolic acidosis in the neonatal period. A
physical examination at 8 months showed failure to thrive, hypotonia, small muscle mass,
severe head lad, and a persistent acidosis, pH 7 to 7.2. Blood lactate (9 mmol/L), pyruvate
(2.4 mmol/L), and alanine (1.36 mmol/L) were greatly elevated. Since these symptoms
suggested a genetic defect in pyruvate metabolism (Blass, 1983), treatment with thiamine,
biotin, bicarbonate, protein restriction, and a ketogenic diet were all tried, but none of the
treatments alleviated the lactic acidosis. After informed consent, oral lipoic acid (25 to 50
mg/kg of body weight) greatly improved the lactic and pyruvic acidemia. The patient was
doing well two vears later.
Questions relating to case:
1. Why were the plasma concentration of pyruvate, lactate, and alanine

abnormally high?
2. Enzyme activity of the PDH complex, a-ketoglutarate dehydrogenase

complex, and dihydrolipoyl dehydrogenase from sonicated skin fibroblasts
grown in culture were all low when compared with enzymes from normal
fibroblasts. Dihydrolipoyl dehydrogenase was especially low. Explain how
a defect in the gene for a single enzyme would lead to these findings in
vivo.
3. Lipoic acid added to the fibroblast homogenates did not stimulate

dihydrolipoyl dehydrogenase activity, yet lipoic acid had a dramatic effect in
vivo. Can you explain this?
4. What was the rationale for attempting therapy with thiamine, biotin,
bicarbonate, and protein restriction?
5. Restriction of dietary carbohydrate to 40% of the food calories has been

fairly effective in alleviating the symptoms of lactic acidosis in patients in
whom the genetic defect in the PDH complex is presumably in the Ei
subcomplex. In this patient, it caused a worsening of the acidosis.
Suggest an explanation.
CASE DISCUSSION:
Genetic defects in pyruvate metabolism may affect either the PDH complex or pyruvate
carboxylase, both mitochondrial enzymes. The patient in this case had an inborn error
in the gene for one of the enzymes of the PDH complex. Because this complex is so
large, containing three enzymatic proteins and five coenzymes, the assay of its
component parts in clinical material is subject to various technical difficulties (Stansbie
D et. al., 1986). It is especially difficult to determine with certainty that the Ei complex is
defective, despite several independent reports of patients who presumably had
defective genes for Eithe PDH complex (Blass, 1983). It may be easier to identify
defects in E2 or E3. Virtually all the patients with disorders of the PDH complex show a
lactic acidosis and abnormalities of the nervous system. This is not surprising, since the
brain depends more on carbohydrate utilization for energy and biosynthesis than other
organs and tissues.
Skin fibroblasts in the diagnosis of genetic diseases

It is difficult to determine which tissues are most affected by a genetic defect, and even
more difficult to obtain antemortem tissue samples suitable for biochemical study.
The nuclei of somatic cells contain most of the genetic information of a particular
organism, but in highly differentiated cells the expression of much genetic information is
repressed. Skin biopsies are safe and almost painless. Explants from these can be
grown in tissue culture, where they yield large numbers of fibroblasts. These cells
proliferate rapidly and are sufficiently undifferentiated to be useful in examining many
genetic diseases. Whole fibroblasts or broken cells, as homogenates, are elegant tools
for the study of metabolic processes, particularly when used with labeled substrates.
Because of permeability barriers, one usually cannot assay enzymatic activities in intact
cells; thus the cultured fibroblasts in this case were disrupted by sonication. This
treatment ruptures membranes and exposes enzymes that ordinarily are enclosed by
impermeable membranes. Fibroblasts from this patient oxidized 1-[14C]-pyruvate to
14C 02 at a rate approximately one-third that of normal fibroblasts.
1. High plasma concentrations

The primary defect in this patient is in the PDH complex, which was much less active
than normal. The accumulation of pyruvate in the plasma is consistent with this
defect. Much of the accumulated pyruvate was either reduced to lactate or
transaminated to alanine, accounting for the accumulation of these substances.
2. Gene defect
A defect in a gene for an enzyme common to both the PDH complex and the a-
ketoglutarate dehydrogenase complex would produce these results. The only protein
component in common with both of these a-keto acid dehydrogenases is
dihydrolipoyl dehydrogenase, E3, which had exceptionally low activity. Thus it is likely
that the gene for E3, or a gene that affects the activity of E3, is defective in this
patient. Assuming this to be true, one might predict that a-ketoglutarate and its
metabolites would also accumulate in the patient; this proved to be true.
3. Effect of lipoic acid
Lipoic acid in the PDH complex is covalently attached to E2, not to E3. E2 is
dihydrolipoyl transacetylase. Possibly the lipoate-activating enzyme, the enzyme that
links lipoic acid to E2, is partially defective in its ability to use lipoic acid; for example,
the Km for lipoic acid of the mutant enzyme might be higher than normal. Also one
must assume that in the absence of a complete E2, E3 is unstable and irreversibly
inactivated. High concentrations of lipoic acid might compensate for the increased
Km of the mutant lipoate-activating enzyme and allow transfer of lipoate to E2, which
would then stabilize E3. Lipoate would fail to do this in vitro if an unstable E3 had
already been inactivated in vivo.
4. Treatment rationale
Therapy with thiamine was attempted because if the patient were defective in E1 ,
such that the enzyme affinity for thiamine was reduced, excess thiamine might allow
the mutant enzyme to function. Biotin was tried for a similar reason. Defects in
pyruvate metabolism usually affect either the PDH complex or pyruvate carboxylase,
pyruvate carboxylase, as with many other carboxylases, contains covalently attached
biotin. Thus the rationale for biotin therapy was similar to that for lipoic acid therapy.
Bicarbonate was used in an attempt to lower the lactic acidosis. This might be
reasonable therapy for an acute attack of lactic acidosis, but it is not a very useful
therapy for the persistent acidosis experienced by this patient. Protein restriction was
tried because the branched-chain amino acids (valine, leucine, isoleucine) are
catabolized by a-keto acid dehydrogenase complexes that are similar to pyruvate
and a-ketoglutarate dehydrogenase and, therefore, might be defective in this
disease.
5. Dietary carbohydrate and acidosis

A genetic defect in E1, leading to a defect in the conversion of pyruvate to acetyl
CoA, might be tolerated with a diet low in carbohydrate, which is catabolized primarily
to pyruvate and lactate. The acetyl CoA needed for operation of the Krebs cycle
could be derived from the fatty acids in the lipid-rich diet. In this patient, however, the
defect is in E3, or in a gene that affects E3. Since E3 is a common component of
several a-keto acid dehydrogenases that function in the oxidative decarboxylation of
keto acids derived from lipids or from ketogenic amino acids, it is not possible to
restore activity to the Krebs cycle by simply lowering dietary carbohydrate and
increasing dietary lipids; the a-ketoglutarate dehydrogenase complex remains
blocked.
Matalon R et ai: Lipoamide dehydrogenase deficiency with primary lactic acidosis:

favourable response to treatment with oral lipoic acid, J Pediatr 104:65, 1984.
Mituda S et ai: pyruvate dehydrogenase subcomplex with lipoamide dehydrogenase

deficiency in a patient with lactic acidosis and branched chain ketoaciduria. Clin Chim
Acta 140:59. 1984.
THE TRICARBOXYLIC ACID (TCA) CYCLE
Required Reading:
Wilkins: p. 73-85 Chapter 3. Section: The Tricarboxylic Acid (TCA) Cycle
Suggested Reading:
Ferrier: Chapter 9, II. B. Citrate Synthesis through the end of the chapter
Objectives:
1. Define the process of the TCA cycle.
2. Explain the purpose of the TCA cycle.
3. Identify where the TCA cycle takes place in the cell.
4. Explain how the TCA cycle is carried out in the cell, and how vitamin deficiencies
would affect this pathway.
5. Explain when the TCA cycle takes place.
A. Name and recognize the structures of the starting products, key intermediates, and
the end products of the TCA cycle. (Obj. 1 and 4)
B. Name the 8 enzymes involved in the TCA cycle and describe the reactions they
catalyze. (Obj. 4)
C. Identify the four reactions of the TCA cycle where reducing equivalents are produced.
(Obj. 2 and 4)
D. Identify the products per turn of the TCA cycle, and be able to differentiate why
glucose fuels 2 turns but alanine can only fuel 1 turn of the TCA cycle.
(Obj. 1, 2, 3, and 4)
E. Identify the 3 enzymes that are regulated steps in the TCA cycle; and the regulators
of the first step of the TCA cycle. (Obj. 5)
F. Define the terms: amphibolic and anaplerotic. (Obj. 1 and 2)
G. Describe the role of the TCA cycle in energy production and how intermediates can
be used for other synthetic processes; and how anaplerotic reactions can replenish
TCA cycle intermediates. (Obj. 1, 2, and 3)
H. Explain why thiamine deficiency will cause elevated levels of pyruvate and lactate in
the blood, as well as how it will affect the TCA cycle. (Obj. 2, 3, 4, and 5)
X. THE T R IC A R B O X Y L IC A C ID C Y C LE (TC A ) C YC LE
A. W hat?
B. W hy?
C. W here?
D. F ig u re 3.5, p. 73: The n e t reaction o f the TC A cycle:
E. How?
F ig u re 3.6, p. 75: The TC A cycle

STEP 1Entry of acetyl CoA into the cycle by condensation with
oxaloacetate. (Figure 3.7, p. 76)
Enzyme:
Key Points:
1. Citrate is also known as: ________________
2. The hydrolysis o f_____________pulls the overall reaction far in the

direction of citrate synthesis.
3. Citrate can also be used as the transporter of:
STEP 2Isomerization of citrate to isocitrate. (Figure 3.8, p. 77)
Enzyme:
Key Points:
1. Why must citrate be isomerized?
2. The isomerization involves two steps both catalyzed by

a . ____________ , then
b . ____________
3. Net result:__________________________.
4. Enzyme named after__________________.

STEP 3The first oxidation-reduction reaction of the cycle. (Figure 3.9,
p. 78)
Enzyme:
Key Points:
1. This is the first o f_________redox reactions in the cycle.
2 . ____________________ catalyzes both the oxid/red reaction and

the decarboxylation reaction
3. The intermediate_______________is a n ___________________ .
4 . _______________ is an amino acid precursor; can be converted to

glutamate by a transamination reaction.
STEP 4The second redox reaction of the cycle. Shown as a simplified

two-step reaction sequence (as applies to the Oxidation States Flow
Chart). (Figure 3.10, p. 79)
Enzyme: __________________________
Key Points:
1. This reaction is very___________and____________
2. This is a decarboxylation of an a-keto acid, followed by an oxidation-
reduction reaction
3. Note the product is attached to ______________
4. This enzyme complex also consists o f______catalytic enzymes and
requires similar cofactors as the PDH complex:
Thiamine pyrophosphate (TPP); lipoic acid; FAD; NAD+; and CoA
5. This complex is a regulated step of the TCA cycle. However, it does
not have the kinase and phosphatase regulatory subunits like the PDH
complex.
6 . _____________ is a precursor in heme biosynthesis.
STEP 5A substrate level phosphorylation which produces GTP. (Figure
3.11, p. 80)
Enzyme: ____________________
(a.k.a.: succinate thiokinase)
Key Points:
1. A basic SLP reaction is defined as: ADP + S-P ATP + S
(S-P = compound with a high energy bond, i.e. anhydride or
thioester)
2. Succinyl thioester of succinyl CoA is energy rich bond.
3. Cleavage of thioester bond of succinyl CoA is coupled to
phosphorylation of GDP.
4. This reaction is readily______________
5. Only step in the cycle that directly yields____________________.
6. GTP use for:
a.
b.
c.
The last 3 rxns are to regenerate oxaloacetate (C4) so that the cycle
can continue. No more carbons (CO2) are lo s t
STEP 6The third redox reaction of the cycle. (Figure 3.12, p. 81)
Enzyme: __________________________
Key Points:
1 . _________ nearly always the electron acceptor in alkane to
alkene oxidations.
2. FAD remains_________________to the enzyme.
3. This enzyme part o f_____________of the ETC.
4. Thus the 2 electrons are transferred immediately to the Fe-S clusters
of the complex and used by the ETC.
STEP 7A hydration step to go from an alkene to a secondary alcohol.

(Figure 3.13, p. 82)
Enzyme:
STEP 8The fourth redox reaction and completes the cycle. (Figure 3.14,
p. 82)
Enzyme:
Key Points:
1. This reaction is unfavorable for the formation of OAA.
2. The key to forming OAA is to keep th e _____________in the
mitochondria (i.e. as soon as it is formed, it is used) and the malate
concentration high.
3. The ratio of NADH/NAD+ is key to the regulating the_____________
4. Oxaloacetate is _____allowed to cross the inner mitochondrial
membrane.
5. Malate____cross the inner mitochondrial membrane.
6. When gluconeogenesis is occurring in a cell, oxaloacetate (formed by
pyruvate carboxylase under these conditions) is quickly reduced to
malate for transport to the cytosol. (Again, [OAA] is kept low.)
When?
F. Regulation of the TCA cycle
1. Citrate synthase (step 1)

a. Reaction:
b. Regulation:
Activator:
Inhibitors include:
2. Other regulated enzymes of the TCA cycle

a. Isocitrate dehydrogenase (step 3)
b. a-Ketoglutarate dehydrogenase complex (step 4)
(Note: Metabolite control of this complex is very similar to the
PDH complex, though it is not hormonally controlled because it
does not have the kinase and phosphatase components like the
PDH complex)
G. TCA Cycle as a Provider of Intermediates for Biosynthetic Pathways
1. Examples:
a. Succinyl CoA provides carbon atoms fo r________(hemes)
b. a-ketoglutarate and oxaloacetate are intermediates for
2. Problem: TCA cycle intermediates must be replenished if any

are drawn off for biosynthetic reactions if the cycle needs to
continue.
Solution: Anaplerotic reactions (meaning to fill up) replenish
intermediates. Examples include:
a.
b.
H. ENERGY CONSIDERATIONS
Table 3.2, p. 84: ATP yield from the complete catabolism of a molecule of
glucose.
1 ATP (hydrolysis) = -7.3 kcal/mol

32 ATP (hydrolysis) = -234 kcal/mol
If you were to completely oxidize glucose = -686 kcal/mol
Efficiency:-234/-686 = ~ 34%
ELECTRON TRANSPORT CHAIN AND

OXIDATIVE PHOSPHORYLATION
Required Reading:
Wilkins: p. 87-97 Chapter 4. The Electron Transport Chain
Coursepack: XI. R. Inhibitors of Oxidative Phosphorylation (problem set) and the ETC
Clinical Cases (I, II, and III)
Suggested Reading:
Ferrier: Chapter 6, V. Electron Transport Chain through the end of the chapter
Objectives:
1. Define the process of the electron transport chain (ETC).
2. Explain the purpose of the ETC, including the special function of the ETC in brown
fat.
3. Identify where the ETC takes place in the cell.
4. Explain how the ETC is carried out in the cell and in specialized tissues (i.e. brown
fat).
5. Explain when the ETC takes place.
6. Explain how specific uncouplers and inhibitors would affect the functions of the ETC,
and the general features of mitochondrial neuropathies and myopathies based on the
mitochondrial genome.
A. Identify the 2 types of ETC prosthetic groups that carry both protons and electrons,
and the 3 types of ETC prosthetic groups that only carry electrons. (Obj. 1, 2, and 4)
B. Identify the specific electron donors (substrates) and final electron acceptor
(product) of each complex I through IV. {Obj. 2, 3, and4)
C. Identify which of the complexes (I through IV) is capable of pumping protons.
{Obj. 2, 3, and 4)
D. Describe the chemiosomotic mechanism of oxidative phosphorylation:
1. How is the membrane potential generated, and how is it used?
2. What is the mechanism of uncoupling the membrane potential from ATP
generation, and why would this be necessary?
3. What is the meaning of the term respiratory control?
{Obj. 1, 2, 3, 4, 5, and 6)
E. Identify specific uncouplers and inhibitors of oxidative phosphorylation and explain
their actions on electron transport, oxygen consumption, and ATP synthesis.
{Obj. 2, 3, 4, 5, and 6)
F. Describe the special features of brown fat mitochondria as generators of heat.
{Obj. 2)
G. Explain the general features of mitochondrial neuropathies and myopathies. {Obj. 6)
XI. THE ELECTRON TRANSPORT CHAIN (ETC)
A. What?
B. Why?
C. Where?
D. Overall process:
Figure 4.1, p. 88
Note: Ratio of 3 ATP made for 1 NADH consumed.
Obviously there is plenty of energy to get the job done, but HOW do
we couple these reactions?
E. How?
1. Electron transfer through Complexes l-IV forms a
2. This_________________ is used to drive the synthesis of ATP

at Complex V.
F. Overview of the Electron Transport Chain

1. Metabolic energy from oxidation of food materialssugars, fats, and
amino acidsis funneled into formation of reducing equivalents
NADH and FADH2.
2. Electrons move from NADH and FADH2 to molecular oxygen
forming a membrane potential (~ 200 mV), which is electrical
energy.
3. The membrane potential (electrical energy') is used to drive ATP
synthesis (chemical energy).
Figure 4.2, p. 89
G. How much ATP can NADH and FADH2 generate?
1. To synthesize 1 ATP from ADP, - 7.3 kcal/mol is needed;
therefore, to synthesize 3 ATP, - 22 kcal/mol is needed.
2. NADH generates - 52 kcal/mol
3. You only need to be 40% (22/52) efficient to make this work, as the
rest of the energy is lost as heatwhich is used to maintain body
temperature.
4. FADH2 only generates about - 40 kcal/mol. An approximate 40%
efficiency only allows for 2 ATP to be made from FADH2 .
5. Thus yields are: 3 ATP per NADH; 2 ATP per FADH2 .
H. How does the electron transport chain work?
____________________ : Which suggests that the mitochondria

establishes and utilizes membrane potential as a driving force for
ATP synthesis.
I. General Structure of the ETC

1. Complex l-IV components:
a. A series o f___________and____________ membrane proteins
with electron and proton carrying groups.
b. These numerous_________________accept electrons from
NADH and FADH2 and pass them through the complexes l-IV to
ultimately donate them to _________________to form ________ .
c . __________ are pumped into the inner membrane space from
the matrix forming a proton gradient as the electrons are moved
through complexes l-IV.
2. Complex V: a n ___________membrane protein complex that

synthesizes ATP from ADP + Pi by allowing the protons to leak back
from the intermembrane space into the matrix.
Figure 4.3, p. 90, The electron transport chain

J. The prosthetic groups of the ETC2 major categories:
1. Category 1: Carriers of both protons (H+) and electrons (e )
[i.e. full hydrogen atoms]
a. Flavin mononucleotide (FMN - FMNFh) in Complex I
[and FAD - FADFh does the same job in Complex II]
Figure 4.4, p. 91: FMN and FMNH2
b. Coenzyme Q (ubiquinone) is reduced to ubiquinol (C0 QH2 )

(i) Has a very___________tail (isoprene units)
(ii) Coenzyme Q - is a highly mobile electron carrier; it shuttles
electrons from Complexes I and II to Complex III
Figure 4.5, p. 92: Oxidized and reduced structures of CoQ.
J. The prosthetic groups of the ETC2 major categories (continued ):

2. Category 2: Carry ONLY electrons
a. Heme (iron-protoporphyrin IX)
(i) Prosthetic groups o f___________(as well as Hb and Mb)
(ii) Cytochromes are: _________with electron transfer functions.
(iii) The iron (Fe) atoms of these hemes only carry electrons,
which alternate between the reduced ferrous state (+2) and the
oxidized ferric state (+3) during electron transport.
Cytochromes c: is soluble (carries e- between complexes III

and IV)
Cytochromes b and C1 are in Complex III
Cytochromes a and a3 are in Complex IV
b. Iron-sulfur (Fe-S) clustersare in Complexes I, II, and III
Iron atom only carries electrons, again alternates
between the reduced ferrous state (+2) and the oxidized ferric
state (+3) during electron transport.
c. Copper (Cu) ions C u a and C u b located in Complex IV
The metal atomcopperonly carries electrons.
K. COMPLEX I:
1. Other names for complex:
2. Electron donor to complex I:
3. Final Electron acceptor from Complex I:
4. Are protons pumped by this complex?
L. COMPLEX II:

M. COMPLEX III:
N. COMPLEX IV:
5. Molecular oxygen (O2): Ideal terminal acceptor because

a. Its high affinity for electrons provides a large thermodynamic
driving force for oxidative phosphorylation
b. O2 also reacts slowly unless activated by a catalyst
6. Reduction of O2 can be dangerous because partial reduction

generates hazardous compounds (termed Reactive Oxygen Species
or ROS)-i.e. superoxide anion (O2'), which is formed by a one
electron transfer to oxygen.
O. COMPLEX V: ATP SYNTHASE [aka. F0Fi-ATPase]
1. Oxidative Phosphorylation: The proton gradient (i.e. membrane
potential) generated to this point by oxidation reactions is now
coupled to phosphorylation of ADP to form ATP.
2. Net Reaction: Membrane Potential (~ 200 mV) + ADP + Pi - ATP
3. ATP synthase has 2 units: __
a. Fi unit (subunit is in the mito. matrix): F0
function: -------- --------
b. Fo unit:
function:
Fi
(J. LaPres, MSU)
4. How does H+ flow drive ATP synthesis? Boyer and Walker, 1997
chemistry Nobel Prize work.
P. Regulation of the ETC

1 . _____________ reactions usually are potential regulatory points.
2. Path from _________to __________ functions nearly at equilibrium
(i.e. is readily reversible)
3 . ___________________________reactionirreversible
a. Controlled by availability of its electron donor:
____________cytochrome c (Fe2+)
b. Since cytochrome c is in equilibrium with the rest of the ETC,
its concentration depends on the intramitochondrial ratios of
[NADH]/[NAD+] and [ATP]/[ADP][Pi] (i.e. the energy state of the
cell)
c. Inactive person (at rest):
d. Active person:
Q. Coupled versus Uncoupled Mitochondria
1. Coupled Mitochondria
a. Electron transfer (and oxygen consumption) through Complexes
l-IV results in ATP synthesis at Complex V.
b. If ATP synthesis stops (because cell has enough for now), then
electron transfer (and oxygen consumption) through Complexes
I-IV also stops.
c. Electron transfer through Complexes l-IV and ATP synthesis at
Complex control each othertermed respiratory control.
2. Respiratory Control
a. The generation of a proton gradient in the intermembrane space
by electron transfer through Complexes l-IV can only build up to a
certain level.
b. Thus, unless there is an active process depleting the proton
gradient (i.e. ATP synthesis or uncoupling processes)electron
transfer will ultimately be inhibitedand oxygen consumption will
stop as well.
c. Physiologically critical to have product inhibition of electron
transfer when the proton gradient is building up too high, without it
being depleted.
d. Mitochondrial function can be measured using the membrane
potential:
The amount of ATP made per oxygen consumed is termed:
P/0 ratio
For NADH the P/0 ratio is 3 (i.e. 3 ATP per O2 consumed)
For FADH2 the P/0 ratio is 2 (i.e. 2 ATP per O2 consumed)
3. Uncoupled Mitochondria
a. Electron transfer occurs through Complexes l-IV, along with
oxygen consumption, without ATP synthesis.
b. In this case heat is produced, which is one of the physiological
roles of mitochondria in specialized tissue called brown fat.
c. Mechanism: dissipate the proton gradient by making
mitochondrial membranes leaky to protons (using specialized
channels or uncoupling agents)
Typically are compounds that are H+ acceptors and that are
lipophilic so they can go across the mitochondrial membranes.
BMB 515
A T P P R O D U C TIO N : C o u p le d M ito c h o n d ria
(Ferrier7_F6.13)
MITOCHONDRION
Inner membrane
I 0uter membrane ADP + Pi
ATP
Complex V
(F 1 domain)
Intermembrane
Matrix ^ space
ATP/ADP
NADH transporter
M IT O C H O N D R IA L '
M A TR IX
Complex V
(F0 domain)
Q
IN T E R M E M B R A N E
CytC
S PA C E
ADP
ATP
H E A T P R O D U C TIO N : U n c o u p le d M ito c h o n d ria (see Brown fat, next page)
(J. LaPres, MSU)
in h ib itio n
Heat p ro d u c tio n : c o n tro lle d by n o re p in e p h rin e via a

cA M P -dependent lipa se
3. Uncoupled Mitochondria (continued)
d. Brown fat (aka. brown adipose tissue or BAT): specialized form
of adipose tissue designed for controlled heat production.
(i) Present in newborns and hibernating animals (very little in
adults)
(ii) Mitochondria of brown fat contain thermogenin (UCP1 or
uncoupler protein 1), an inner membrane proton channel.
(iii) Thermogenin acts as a regulated uncouplerwhen the
channel is open, it functions to short circuit the respiratory chain
and leaks protons back into the matrix.
(iv) Thus no work (i.e. ATP synthesis) is done, but heat is
produced.
(v) In adults, BAT does not control heat production. However,
heat production may be controlled by uncoupling proteins 2 and
3 (UCP2,UCP3)
(vi) UCP2 is also known as an obesity gene.
ETC INHIBITORS (J. LaPres, MSU)

AMYTAL
ADP + Pi
2,4-DNP IS AN UNCOUPLER
R. Inhibitors of Oxidative Phosphorylation
Inhibitors of Electron Inhibitors of Uncouplers

Transport Phosphorylation
Complex I: Rotenone, Amytal Complex V: a. 2,4-dinitrophenol
Complex III: Antimycin Oligomycin (2,4-DNP)
Complex IV:
Cyanide (CN ) b. Free fatty acids
Azide (N3 ) (natural)
Carbon monoxide (CO)
Hydrogen Sulfide (HS)
Effect: Block electron Effect: Blocks Effect: Electron
transport & phosphorylation phosphorylation & transport
(because are coupled) electron transport occurs WITHOUT
(because are phosphorylation
coupled)
1. So if any of the Complexes I, III or IV are inhibited, the effects are:

Electron transport _________
Oxygen consumption _________
ATP synthesis _________
Effect of uncoupler addition? _________
What ETC intermediates will build up/decrease? _________
2. Oligomycin inhibits the ATP synthase; the effects are:

Effect of uncoupler addition? _________
3. Uncouplers uncouple oxidation and phosphorylation; the effects are:

ETC Clinical Case I:
A patient is brought into the emergency room on the verge of death. He

was exposed to a mitochondrial poison, but you do not know which one.
You know that he is not making enough ATP and his mitochondrial oxygen
consumption is very low. Using virtual technology you are able to deduce
that the addition of 2,4-DNP to his mitochondria does not rescue oxygen
consumption. Finally, these purified mitochondria have an excess of
reduced cytochrome c. To which of the following poisons was the patient
exposed?
A) Amytal
B) Rotenone It is a binary decision!
C) Oligomycin You know the patient is not making ATP,
D) Cyanide so you ask yourself the following questions:
Are the mitochondria still consuming oxygen?
YES X \ NO
/ \
Does 2,4-DNP addition
restore oxygen consumption?
YES
Y
Is there excess NADH
and absence of QH2?
NO
,k / \
Is there excess QH2 and
absence of reduced Cyt. C?
\ NO
Is there excess of
reduced Cyt. C?
YES
I
REQUIRED READINGS (FOLLOWING 2 CLINICAL CASES)
ETC Clinical Case II:
lultiple Deletions o f M itochondrial DNA in Several Tissues of a Patient with

Severe R etarded D epression and Familial Progressive External O phthalm oplegia
Anu Suom alainen,* Anna M a jan d er,* M a tti H altia,* H annu S am er,u Jouko Lonnqvist ,1
Marja-Liisa S avo ntaus,** an d Leena P elto nen*
*Department o f Human Molecular Genetics, National Public Health Institute. 00300 Helsinki: *Department o f Medical Chemistry,
University o f Helsinki. 00170 Helsinki: Department o f Pathology, University o f Helsinki. 00300 Helsinki; *Department o f Neurology
University o f Helsinki. 00290 Helsinki: 'Department o f Mental Health. National Public Health Institute. 00300 Helsinki:
and **Department o f Medical Genetics. University o f Turku. 20520 Turku. Finland
A bstract are, however, encoded by nuclear genes. Consequently a muta

tion in either genome may result in mitochondrial dysfunction.
Multiple deletions of mitochondrial DNA (mtDNA) have re
cently been reported in familial progressive external ophthal Mutations o f mtDNA have been associated with several
moplegia (PEO), in a case of progressive encephalomyopathy, human diseases ranging from mild myopathies to severe multi
and in inherited recurrent myoglobinuria. The inheritance of system disorders. Large single deletions were first reported in
familial PEO has been autosomal dominant, which indicates mitochondrial myopathies (I) and have been later shown to
that a mutation in an unknown nuclear gene results in several exist in progressive external ophthalmoplegia (PEO) (2, 3),
mtDNA deletions o f different sizes in these patients. We report Kcams-Sayre syndrome (3, 4), and Pearsons syndrome (5).
a patient with autosomal dominant PEO, whose major clinical Multiple deletions o f mtDNA have been reported in familial
symptom, however, was severe retarded depression. The mor PEO (6-8), familial mitochondrial myopathy (9), a case o f pro
phological analyses of the tissue samples derived from autopsy gressive encephalomyopathy (10), and in inherited recurrent
showed various abnormalities in the mitochondria in all the myoglobinuria (II).
tissues studied. The activities of the respiratory chain enzymes MtDNA is inherited through the maternal lineage. How
tided by mtDNA were remarkably reduced in the skeletal ever, only the diseases associated with point mutations of
uscle. The mtDNA analyses confirmed that besides myopa mtDNA show matrilinear transmission. Single deletions are
thy, this patient had a multisystem disorder with widespread mostly sporadic with variable tissue involvement, which sug
distribution of multiple deletions of mtDNA. The highest per gests the mutations to be o f somatic origin. However, some
centage of mutated mtDNA was found in the brain, skeletal reports of maternally inherited single deletions o f mtDNA exist
muscle and the heart, the relative quantity of mutated mtDNA in the literature (2, 12). The PEO families (6-8) with multiple
correlating to the severity of the clinical symptoms. ( / . Clin. deletions o f different sizes show an autosomal dom inant mode
Invest. 1992. 90:61-66.) Key words: encephalopathy myopa o f transmission. This could be explained by a factor encoded by
thy oxidative phosphorylation respiratory chain slip replica nuclear genes that somehow disturbs the interaction between
tion the nucleus and mitochondria resulting in multiple mtDNA
deletions in these patients (6-8).
Introduction To date, multiple deletions o f mtDNA have been found
only in the muscle (6-8) o f the patients with autosomal domi
H ie mitochondria are ruled by two different genomes, the nu nant PEO. We report here molecular genetic, morphological,
clear and the mitochondrial. The mitochondrial genotype is and biochemical studies o f an autopsied patient who suffered
the result of several thousand mitochondrial DNA (mtDNA)1 from autosomal dominant PEO and major depressive episodes
copies in each ceil. Human m tpN A is a circular double- with severe psychomotor retardation. Multiple deletions of
stranded molecule of 16.6 kb. It encodes 13 subunits o f the mtDNA were found in several tissues analyzed, the most se
respiratory chain enzymes, as well as the mitochondrial ribo- verely affected tissues being the brain, heart, and the skeletal
somal and transfer RNAs. M ost o f the mitochondrial proteins muscle.
CARBOHYDRATE METABOLISM:
Mitochondrial Electron Transfer/Oxidative Phosphorylation
Case report
The patient was the seventh of ten children of unrelated parents (Fig. 1). Her
development was normal in childhood. From the age of 19 yr onward she suffered from
successive episodes of severe psychomotor retardation and depressive mood with apathy,
mutism, fatigue, insomnia, and negativism as well as loss of initiative and appetite, fulfilling the
criteria of major depressive disorder (DSM-III-R). The depression was often accompanied by
long periods of amenorrhoea. The patient was treated with electric and insulin shocks as well
as with antidepressive drugs. Between the depressive episodes the patient worked as a
housemaid and was considered healthy by her siblings. After the age of 50 yr the depression
changed to a more chronic stage, and she became unable to work.
In the physical examination at the age of 52 yr the patients height was 148 cm and
weight 32 kg. She had bilateral ptosis, first detected at the age of 29 yr, and limitation of eye
movements in all directions. She had generalized muscle weakness corresponding to the
reduction of muscle bulk. The histochemical and electron microscopic study of a muscle biopsy
specimen established the diagnosis of mitochondrial myopathy.
At the age of 60 yr the patient was admitted to hospital because of chest pain and
dyspnoea. She was cachectic and suffered from delusions and paranoia. Serum creatine kinase
(CK) value was 1.962 U/liter (normal < 150 U/liter) of which CK-MB comprised 12% and CK-
BB 7%. Serum lactate dehydrogenase activity was 1.051 U/liter with an increase predominantly
in the lactate dehydrogenase 1 fraction. The enzyme activities returned to normal within a few
days, but the CK-MB and -BB isoenzymes remained detectable. However, repeated
electrocardiogram examinations did not show any signs of myocardial ischemia. In 1 wk the
patient was resuscitated twice from ventricular fibrillation; then she became comatose and died.
The patients mother had progressive ptosis, and four of the siblings and three of their offspring
suffer from PEO and muscle weakness of respiratory muscles and is dependent on assisted
ventilation. Although the psychiatric examination of the family members has not yet been
completed, a tendency to depressive mood, e.g., suicide attempts and withdrawal from the
community, has been reported in the affected individuals. The proband was the only one
receiving psychiatric treatment.
excerpted from J. Clinical Invest. July (1992)
1. Knowing this is a mitochondrial disease, would you expect lactic acidosis (as in PDH
case)?
2. Why might the disease be episodic and progressive with age? (Refer to next page.)
Highlights of Introductory material from the J. Clinical Invest report:
The authors point out that the mitochondria is ruled by two genomes.
Mutations in the mitochondrial DNA (mtDNA) have been associated with
various diseases ranging from mild myopathies to severe multisystem
disorders.
mtDNA is inherited maternally; however, only point mutations show true
matrilinear transmission.
Deletions in the mtDNA are sporadic and vary in tissues suggesting
somatic origins.
The patient from the case showed multiple deletions in her mtDNA and in
multiple tissues (most severly affected were brain, heart, and skeletal
muscle).
Question #1: Knowing this is a mitochondrial disease, would you expect

lactic acidosis (as seen in the PDH case)?
The patient would not necessarily show lactic acidosis because the
liver is not one of the organs severely affected by the mitochondrial defect.
Therefore, under most conditions, the liver would be able to clear the lactic
acid produced due to mitochondrial malfunctions in other tissues.
Question #2: Why might this disease be episodic and progressive with
age?
The disease may be episodic because the level of mitochondrial

function is barely adequate under normal conditions and increase activity or
stress may push the system beyond its capacity, causing tissue
malfunction and damage. Some damage may be repairable over time,
leading to recovery.
The disease is likely to be progressive with age due to the continual

decline in the number of active mitochondria with aae.
ETC Clinical Case III:
Cytochrome Oxidase Inhibition Case
A 26-year-old man without previous past medical history was working on a natural gas well. The
oil well appeared to have a leak surrounding the main chamber, and as the patient bent forward to fix
the gas leakage, he reportedly lost consciousness and suffered a cardiopulmonary arrest. Emergency
medical services was called to the scene, and the patient was found to be apneic and CPR was
performed briefly. He then resumed spontaneous respirations, as well as normal blood pressure, and
was taken to an outside emergency room, where he was found to be confused, disoriented and
hypoxic. He was placed on a 100% non-rebreather face mask and was found to have arterial blood
gas with a pH of 7.33, pC03 of 43, and a p02of 193. Chest x-ray at the time demonstrated diffuse
bilateral infiltrates throughout all lung fields. After the patient was stabilized, he was then transferred
to a tertiary care hospital.
He was a non-smoker, took no medication, occasionally drank alcohol and worked for 6 years at a
gas well drilling company.
Laboratory Data: White blood cell count 13.9, hematocrit 43.2, BUN 22, and creatinine 1.0. Cardiac
enzymes were within normal limits. Follow-up arterial blood gas on room air revealed t pH of 7.43,
pC02of 34, p02of 52, with a saturation of 87%. Methemoglobin was 1.2 with a carboxyhemoglobin
of 1 1. EKG revealed sinus tachycardia at a rate of 108, nonspecific T-wave changes (for normal
values see Case #1 Course Pack).
Hospital Course:
1 Pulmonary: The patient initially was supported with oxygen by nasal cannula, which was
subsequently weaned off over the next couple of days. The patients chest x-ray continued to
resolve with evidence of decreased air space opacity, without evidence of superimposed
aspiration pneumonia and remained without cough.
2. Cardiac: The patient initially was admitted to a monitored bed given his history of possible
cardiac arrest. He remained in a normal sinus rhythm throughout his hospitalization without
evidence of ectopy or other arrhythmia.
3. Ophthalmology: An Ophthalmology consult was obtained given the patients marked scleral
hemorrhage. It was their impression that he had subconjunctival hemorrhages secondary to
hydrogen sulfide exposure, however, no specific ocular treatment was indicated at the time of
the consult. The patient was informed of a possible toxic optic neuropathy, and the patient was
to inform his physician should he develop any decrement in visual acuity.
Hydrogen Sulfide i/a rapidly acting toxin causing respiratory paralysis by inhibition of cytochrome
oxidase. At high concentrations (1000 - 2000 parts per million) coma can occur after a single breath
and death shortly after. Hydrogen sulfide is the smell of rotten eggs you may associate with sewage.
It can be a contaminant in natural gas and oil. Petroleum workers, sewage workers and farmers with
manure pits are all at risk of exposure. This patient developed pulmonary edema (accumulation of
fluid in alveoli) and all the oxygen values in his blood are reduced. There was no evidence of a heart
attack (normal heart enzymes and no EKG evidence of a heart attack). There is no specific treatment
to reverse hydrogen sulfides effect on cytochrome oxidase. Treatment is supportive, intubation and
ventilation, oxygen and treatment of any superimposed infections.
THREE KEY BRANCH POINTS IN METABOLISM
Glucose 6-phosphate (glc 6-P) sits at an important branch point in metabolism. Glucose
(from blood) must pass through glc 6-P to get down to pyruvate in glycolysis. Conversely,
gluconeogenesis (GNG) requires a special enzyme, glucose 6-phosphatase, to convert
glc 6-P to glucose. Glucose 6-P also serves as the starting point for glycogen synthesis
in the temporary storage of glucose energy units. When glycogen is broken down in times
of energy need, glc 6-P represents the end point at which a decision must be made with
respect to the ultimate fate: export to buffer blood glucose concentration (liver, because
it has glucose 6-phosphatase) or utilization in generating A TP (muscle). Another pathway
that starts with glc 6-P is the pentose phosphate pathway (PPP), also known as the
hexose monophosphate shunt (HMP).
Pyruvate is also a key branch point, as has been discussed. The presence or absence
of molecular oxygen determines whether or not pyruvate can move into the mitochondria.
If it can, pyruvate can be used to make oxaloacetate (for gluconeogenesis) or acetyl CoA.
If pyruvate remains in the cytosol, it can be used to make lactate (anaerobic glycolysis)
or alanine.
Acetyl CoA is another key branch point. Acetyl CoA can be used by the TCA cycle or it
can be shuttled back to the cytosol for the synthesis of fatty acids or cholesterol.
Required reading:
Wilkins: p. 99-101, Chapter 5, Section: Three Key Branch Points in Metabolism
Objectives:
1. Define the roles of glucose 6-phosphate, pyruvate and acetyl CoA as key branch
points in metabolism.
XII. T h re e ke y b ra n c h p o in ts in m e ta b o lis m
F igure 5.1, p. 100
g lu c o s e
g ly c o g e n g lu c o s e 6-P rib o s e 5-P
a la n in e ^ p y ru v a te ^ la c ta te
i
a c e ty l C o A
GLUCONEOGENESIS: Self-Studv Application
**Use the REQUIRED On-Line Module and the

Textbooks and Course Pack Reading Assignments**
Required activity and readings:

On-line Self Study Application on Gluconeogenesis (directions on next page)
Wilkins: p. 100-108, Chapter 5: Section on Gluconeogenesis
CoursepackGluconeogenesis: Clinical Case
Ferrier: Chapter 23: IV. Hypoglycemia C. Types 4. Alcohol-related hypoglycemia
Suggested reading:
Ferrier: Chapter 10, entire chapter
Objectives:
1. Define the process of gluconeogenesis.
2. Explain the purpose of the gluconeogenesis, including its clinical relevance in young
children.
3. Identify where gluconeogenesis takes place in the cell, and which tissues/organs can
release free glucose into the blood.
4. Explain how gluconeogenesis is carried out in the cell.
5. Explain when gluconeogenesis takes place.
A. Identify the 3 irreversible reactions of glycolysis, which must be bypassed in

gluconeogenesis. ( Obj. 4)
B. Identify the sources of pyruvate when it is used in gluconeogenesis. (Obj. 1, 2, 3,
and 4)
C. Describe all of the necessary reactions and transports needed in gluconeogenesis to
bypass the 3 irreversible reactions of glycolysis; and know where in the cell these
steps occur. (Obj. 2, 3 and 4)
D. Identify the regulatory steps in gluconeogenesis, the enzymes that catalyze these
steps, and the molecules (compare/contrast with glycolysis) that regulate them.
(Obj. 5)
E. Explain how the reciprocal regulation of glycolysis and gluconeogenesis is
accomplished and why it is important. (Obj. 4 and 5)
F. Identify the physiological circumstances under which gluconeogenesis would be
stimulated and explain why it is stimulated under these conditions. (Obj. 3, 4, and 5)
G. Explain how alcohol affects gluconeogenesis and how this would cause
hypoglycemia. Explain why alcohol consumption would be especially dangerous for
young children. (Obj. 2 and 3)
GLUCONEOGENESIS: SELF STUDY ON-LINE MODULE
Gluconeogenesis is a metabolic pathway that creates glucose from other carbon

sources, such as pyruvate, lactate, glucogenic amino acids, and glycerol.
Gluconeogenesis involves many of the same enzymatic steps as glycolysis. An
online, self study exercise has been created for you to become familiar with the
gluconeogenic pathway. Given the similarities between glycolysis and
gluconeogenesis, it is highly recommended you go through this tutorial as soon
as possible, while the information is still fresh in your mind. Once the tutorial is
completed, you will receive a metabolic cheat sheet to help you review the
process.
Directions:
Go to the BMB 515 D2L website (d2l.msu.edu)
The self study module can be found under the Content tab in the
Tutorials and Gluconeogenesis Self-Study Module folder in a
subfolder entitle REQUIRED: Gluconeogenesis Self-Study Module.
Click on the Gluconeogenesis Self-learning exercise.
Please make sure you read everything.
DO NOT use your browser back arrow/button to try to navigate

through the module once you have started. Because of the way the
tutorial was programmed, this will send you back to the very first
slide.
Follow the instructions within the on-line module.

G LU C O N E O G E N E S IS : R EQ U IR ED R EAD IN G
(W ilk in s , p. 100-108, C h a p te r 5: S e c tio n o n G lu c o n e o g e n e s is )
XIII. GLUCONEOGENESIS (GNG)

A. What?
B. Why?
C. Where?
D. Net reaction: Figure 5.2, p. 102
E. How?
F. When? (i.e. Regulation of Gluconeogenesis)

G. Gluconeoqenesis: Clinical Case
Hypoglycemia and Alcohol Intoxication

(Required Reading: F e rrier, A lc o h o l-re la te d h y p o g ly c e m ia )
Consumption of alcohol, especially by an undernourished person, can cause

hypoglycemia. The same effect can result from drinking alcohol after strenuous exercise.
In both cases the hypoglycemia results from the inhibitory effects of alcohol on the hepatic
gluconeogenesis, and thus occurs under circumstances of hepatic glycogen depletion.
The problem is caused by a massive increase in cytosolic NADH produced during

metabolism of alcohol. The liver simply cannot handle the reducing equivalents provided
by ethanol oxidation fast enough to prevent metabolic derangement. The extra reducing
equivalents block the conversion of lactate to pyruvate to glucose and promote
conversion of alanine into lactate, resulting in considerable accumulation of lactate
accumulation in the blood. Since lactate has no place to go, lactic acidosis can develop,
although it is usually mild. Oxaloacetate reduction to malate is also favored. Thus, the
lack of pyruvate and oxaloacetate (both gluconeogenic intermediates) in the cytosol
impairs gluconeogenesis in the liver.
Low doses of alcohol cause impaired motor and intellectual performance; high doses
have depressant effects that can lead to stupor and anesthesia. Low blood sugar can
contribute to these undesirable effects of alcohol. What is more, a patient may be thought
to be inebriated when in fact the patient is suffering from hypoglycemia that may lead to
irreversible damage to the central nervous system.
Children are highly dependent upon gluconeogenesis while fasting and accidental
ingestion of alcohol by a child can produce severe hypoglycemia.
Krebs, H.A., Freedland, R.A., Hems, R., and Stubbs, M.

Inhibition of hepatic gluconeogenesis by ethanol.
Biochem. J. 112: 117, 1969
NADH NADH
NAD+ NAD+
O 0
II
CH3 CH2 OH CH3 C H V . C H C O'
alcohol aldehyde
ethanol dehydrogenase acetaldehyde acetate
dehydrogenase
(C. Wilkins, MSU)

THE PENTOSE PHOSPHATE PATHWAY

(a.k.a. the hexosemonophosphate shunt)
We now discuss the pentose phosphate pathway (PPP), also known as the hexose
monophosphate shunt (HMP), which is another pathway that starts with glc 6-P. This pathway
is important in producing NADPH and ribose 5-P. Because ribose 5-P is the source of carbon
skeleton for the nucleic acids (DNA and RNA), the PPP thus serves as a connection between
carbohydrate metabolism and nucleic acid metabolism. As we shall discuss, the PPP also
produces fructose 6-P and glyceraldehyde 3-P, two intermediates of glycolysis. The importance
of NADPH in various functions of the cell will also be discussed.
Required reading and problem set:

Wilkins: p. 108-121 Chapter 5, Section: The Pentose Phosphate Pathway
Wilkins: p. 122-129 Chapter 5, Problem Set
Suggested reading:
Ferrier: Chapter 13, entire chapter
Objectives:
1. Define the process of the pentose phosphate pathway (PPP).
2. Explain the purpose of the PPP, and identify the uses of NADPH.
3. Identify where the PPP takes place in the cell.
4. Explain how the PPP is carried out in the cellcomparing and contrasting its 4
modes.
5. Explain when the PPP occurs and how it is integrated with glycolysis and
gluconeogenesis.
6. Explain how certain vitamin deficiencies would affect the PPP.
A. Describe the purpose of the pentose phosphate pathway (PPP): to produce NADPH,
to provide ribose 5-P for nucleotide synthesis, and to provide glycolytic intermediates.
{Obj. 1, 2, and 3)
B. Identify the substrates and products of the reactions in PPP catalyzed by glucose 6-P
dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase. (Obj. 4)
C. Explain how the PPP can generate intermediates of the glycolytic pathway and the
role of transketolase (TK) and transaldolase (TA). Identify TK as a thiamine-
dependent enzyme and describe the clinical consequences of thiamine deficiency.
{Obj. 5 and 6)
D. Starting with glucose 6-P, diagram how the reactions of glycolysis and PPP can be
combined to satisfy the need(s) of a cell: (a) both NADPH and ribose 5-P; (b) NADPH
but not ribose 5-P; (c) NADPH and ATP energy; and (d) ribose 5-P but not NADPH.
Give an example of a cell that might run a specific mode of the PPP cited above.
(Obj. 2, 4 and 5)
E. List the uses of NADPH, including its role in generating Reactive Oxygen Species
(ROS) in Respiratory Burst and in detoxifying ROS through glutathione. (Obj. 2)
F. Distinguish the mitochondrial versus the microsomal Cytochrome P450 system in

terms of their respective activities. (Obj. 2)
BMB515
6- P G l u c o n a t e G ly c o g e n G a la c to s e
^
R ib u lo s e 5 - P 6- P g lu c o n o la c to n e
/ UN
I O P -G lu c o s e
I
G a la c to s e 1 -P
R ib o s e 5 -P
//
/ /
*/
X\ v , / -----------------
G lu c o s e 1 -P
\ it
* X i
U D P -G a la c to s e
X y lu lo s e 5 - P G l u c o s e 6- P G lu c o s e
it
S e d o h e p tu lo s e 7 - P * F r u c t o s e 6- P F ru c to s e
E ry th ro s e 4 -F ^ 4^
i
G ly c e r a ld e h y d e 3 -P
F r u c to s e 1 ,6 -b is -P
^ _
G ly c e r a ld e h y d e 3 -P
- G ly c e r a ld e h y d e
V r
D ih y d r o x y a c e t o n e - P
it
F r u c to s e 1 -P
c e ra te G ly c e r o l- P G ly c e r o l
a te l
1 t
> -* *T r ia c y Ig ly c e r o l -
ite t 4-
F a t t y a c y l C o A *--------------F a t t y a c i d
ite / /
M a lo n y l C o A
Leu
Phe
Tyr
~7 --r A c e t o a c e t a t e -* ------ < T rp
\N
p -H y d ro x y b u ty ra te
Lys
to
X
a p c itr a te G in
g lu ta ra te X
O
G lu *
P ro
H is
C O , A rg
M e th y lm a lo n y l C o A
T
P r o p io n y l C o A
A c e ty l C o A
F a tty a c y l C o A
(o d d -n u m b e r c a rb o n s )
6-P Gluconate
Ribulose 5-P 6-P Gluconolactone
Ribose 5-P
Xylulose 5-P Glucose 6-P

X it
Sedoheptulose 7-P Fructose 6-P
Eryth
Fructose 1,6-bis-P
$----->-DHAP
Glyceraldehyde 3-P
Glyceraldehyde 3-P
C o p y ri g h t <S> 2 0 1 1 W o Ite rs K l uiAie r H e a Ith | L i p p i n c o t t W i I lia m s & W i Iki n s
Ferrier7 F13.1
XIV. P e n to s e P h o s p h a te P a th w a y [fro m G lc 6-P]
A. W hat?
B. W hy?
C. W here?
D. How? (overview): 8 steps overall
1. First 3 steps:
2. Last 5 steps:
F ig u re 5.10, p. 111: The Pentose Phosphate Pathway

E. Uses of NADPH
1. R eductive biosynthesis
2. Reduction of hydrogen peroxide (H 2 O 2 )

e e e e
> OH h 2o
oxygen superoxide hydrogen hydroxyl water
peroxide radical
a. Reactive O xygen S pecies (ROS) are:
F ig u re 5.9, p. 110: G lu ta th io n e p e ro x id a s e a n d g lu ta th io n e re d u c ta s e
re a c tio n s
c. C ells have a tripeptide reductant,
Reduced form (free SH on Cys), can detoxify H 2 O 2 through

reaction catalyzed b y ___________________
d. The regeneration o f GSH from G -S-S-G re q u ire s _______________
A N D the reducing pow er o f NADPH.
e. Certain cells (e.g. red blood cells) that depend on the PPP fo r
NADPH experience oxidative stress if the enzym e G 6PD is
deficient, particularly when challenged with oxidant drugs (anti-
m alarials; sulfa antibiotics).
Clinical scenario: While filming a movie in an area endemic for malaria, the famous
actor Gino Casetta developed symptoms of anemia (labored breathing, fatigue with mild
exertion). The film director thought it was caused by the anti-malarial drugs Gino was
taking but the producer disagreed because two other actors, Jim Stone and Boris
Mossowski, took the same medication without any ill effects. What do you think?
Dr, W ilkins: m indockc@ m su.edu
3. Cytochrome P450 (CYP) monooxygenase system

(P450 refers to the absorbance at 450 nm of the protein)
a. Reaction catalyzed by this system
R-H + 0 2 + NADPH + H+ R-OH + H20 + NADP+
b. Two CYP systems

(i) mitochondrial system:
(ii) microsomal system:
4. Generation of the Respiratory Burst by phagocytic cells

a. Once it engulfs foreign material Ferrier7_F13.8
(e.g. bacteria), the phagocyte
(neutrophil, monocyte) needs to
kill the foreign microorganism by
producing
b. uses NADPH and O2 to produce

the first ROS, superoxide anion.
This kills the bacteria as well as
generating the other ROSs: H2O2,
OH, and HOCI (hypochlorous
acid)
5. Synthesis of Nitric Oxide (NO)

Figure 5.11, p. 112: The Pentose Phosphate PathwayOxidative Phase
(irreversible)
F. Oxidative Phase:
Two enzymes of our focus:
1. Reaction 1: Glucose 6-P dehydrogenasethe first

oxidation/reduction reaction producing NADPH
Key Points:
a. This step is ____________and highly_____________
b. Enzyme is highly specific fo r___________
c. Enzyme name is exception to the rule
2. Reaction 3: 6-PhosDhoaluconate dehydrogenasethe second

oxidation-reduction reaction producing NADPH
6-phospho-gluconate is first oxidized and then decarboxylated to

form ribulose 5-phosphate.
Key Points:
a. This step is ______________
b. The intermediate 3-keto-6-phosphogluconate has a
c. This enzyme catalyzes both the oxid/red reaction and the

decarboxylation reaction
d. Enzyme name is exception to the rule
Figure 5.12, p. 114: The Pentose Phosphate PathwayNon-oxidative
Phase (reversible)
G. Non-oxidative Phase:
Two enzymes of our focus:
NOTE: In reactions 6, 7, and 8the donor molecule is always the

ketose, and the acceptor molecule is always the aldose.
Figure 5.14, p. 117: Reaction 7Remove 3 carbons from ketose and put
on aldose
Enzyme: transaldolase (TA)
Key Points:
1. This reaction primarily makes a useful
2. Transaldolase moves a n ____

Figure 5.15, p. 118: Reaction 8Remove 2 carbons from ketose and put
on aldose
Enzyme: transketolase (TK)
Key Points:
1. Transketolase (catalyzes both reactions #6 AND #8):
2. Transketolase uses_______________________to move the

carbon units in the form of a keto group.
NOTE the following:

3. Interconversion of sugars by TK and TA in the non-oxidative phase
yields:______________________________
4. These are glycolytic/gluconeogenic intermediates
a. can go down through remainder of glycolysis to yield energy
OR b. can go back up through gluconeogenesis to regenerate
glucose 6-P
5. Because the non-oxidative phase of PPP is reversible, glucose 6-P
can come down in glycolysis to fructose 6-P and glyceraldehyde 3-P
and then back up the PPP to ribose 5-P.
H. The Pentose Phosphate Pathway is a very flexible pathway
1. Utilization of glucose-6-P depends on cells need for:
2. Glucose-6-Pchoice between glycolysis and the PPP depends on

regulation o f____________________________
3. Even if PPP is chosen, the relative cell needs of ribose-5-P and
NADPH (as well as ATP) dictate what portion(s) of the PPP are
chosen.
4. Four principal possibilities (modes) dictate the set of reactions
combined from the PPP and glycolysis (or gluconeogenesis)
Figure 5.16, p. 120: Integration of the pentose phosphate pathway

with glycolysis and gluconeogenesis
Mode 1:
Mode 2:
Mode 3:
Mode 4:
**{Do the problem set at the end o f Wilkins. Chapter 5. d as. 122-129)**
DNA and Chromosome Structure
Chromosomes are the scaffolding that organizes all of our genetic materials, and allows
for their correct expression, as well as distribution from generation to generation. They are rod
shaped condensations of nucleic material (DNA) formed during cell division. The number of
chromosomes is species specific, and in humans, every somatic cell essentially contains a set of 46
chromosomes, each encompassing a pair of each of the 23 chromosomes. 23 are derived from our
mother, and 23 from our father. We are a diploid organism. Sex chromosomes are identical in the
female (XX) and different (XY) in the male. Every nucleated cell in the body carries its own
copy of the human genome.
In this course, we will use the term gene to define the conceptual bits of hereditary
information, represented materially in the form of DNA. Genetics describes the identification of
those units and how those units interact. Molecular biology describes the mechanisms used to
express that information during the life of an organism and the mechanisms used to transmit that
information to subsequent generations.
In this lecture we will define the flow of genetic information as our guiding principle and will
outline the basic processes at work in molecular biology. We will then cover the structure of DNA
and genomics.
Suggested Reading:
Ferrier: Chapter 30: DNA Structure, Replication, and Repair - from Overview through DNA
Structure and Eukaryotic DNA Organization
Turnpenny and Ellard: Chapter 2: The Cellular and Molecular Basis of Inheritance - DNA: The
Hereditary MateriaD - from Composition through Structure (p. 13-14); from Chromosome
Structure through Types o f DNA Sequence (p. 15-18); and The Genetic Code (p. 20-21)
Optional Suggested Videos:
https://www.voutube.com/watch?v=gbSIBhFw04s [How DNA is packaged]
Objectives:
1. Understand how the genetic information is used through the processes of replication, transcription
and translation (central dogma, degeneracy, unambiguity).
2. Describe the physical structure of DNA and the hierarchical relationship between DNA,
chromatin and chromosome (genome, phosphodiester bond, nucleosome, histone, haploid,
diploid, homologs, sister chromatids, telomere, centromere vs. kinetochore, P-arm and Q-arm,
heterochromatin, euchromatin).
3. Given the organization of the genome, define the informational content and function of the
different types of sequences (genes, regulatory sequences, spacer DNA, microsatellite,
minisatellite).
M e a s u r a b le O u tc o m e s: W h a t y o u sh o u ld b e a b le to d o ...
I. F lo w o f G e n e tic I n fo r m a tio n
1. Distinguish between gene as conceptual unit of information and DNA as the physical
material in which that information is encoded. (Obj. 1 and Obj. 2)
2. Describe the central dogma of molecular biology, including the informational roles of
DNA, RNA and protein. (Obj. 1 and Obj. 2)
3. Define the processes of replication, transcription and translation. (Obj. 1)
4. Define the terms genome and chromosome. (Obj. 2)
5. Describe the principal attributes of the genetic code. Distinguish between degeneracy and
un-ambiguitv in the genetic code. (Obj. 1)
6. Given a DNA or RNA sequence, predict the corresponding protein sequence. (Obj. 1)
7. Given a protein sequence, identify one or more corresponding DNA or RNA sequences.
(Obj. 1)
II. D N A S tr u c tu r e
1. Describe the chemical bond that joins adjacent nucleotides in a nucleic acid polymer. (Obj.
2)
2. Given a diagram of the chemical structure of a nucleic acid, identify the 5' and 3' ends.
(Obj. 2)
3. Identify and describe the positions of the bases, sugars, and phosphates in the double helix
model of DNA structure. (Obj. 2)
4. Describe the forces that contribute to the stability of the double helix. (Obj. 2)
5. Given the sequence or composition of a single strand of DNA, predict the sequence or
composition of the complementary strand. (Obj. 1 and Obj. 2)
6. Define the components and structure of a nucleosome and the role of histone H I. (Obj. 2)
7. Describe the multiple levels of DNA packaging from nucleosome to chromosome. (Obj. 2)
8. Define the terms haploid and diploid and know how those terms correspond to human
cells. (Obj. 1 and Obj. 2)
9. Define and/or describe the following aspects of chromosome structures: homologs, sister
chromatids, telomere, centromere vs. kinetochore, P-arm and Q-arm. (Obj. 2)
10. Compare and contrast heterochromatin and euchromatin. (Obj. 2)
III. G e n o m ic s
1. Describe the informational content of genomes, including: (Obj. 2 and Obj. 3)
Coding regions
Non-coding regions
2. Identify the different types of sequences found in the human genome. (Obj. 2 and Obj. 3)
3. Describe the possible uses for microsatellites and minisatellites in medicine and the role
microsatellites play in disease. (Obj. 3)
I. Flow of Genetic Information
A. Gene as unit of hereditary information: fMetaphor. Cookbook]
1. Gene: the instructions for making a component of the cell (usually a protein or subunit
of a protein, but sometimes is RNAi.e. rRNA or tRNA)
[Metaphor. Each recipe in a book]
Gene (the unit of information) is physically embodied in DNA

[i.e. the paper and ink of the cookbook]
2. Chromosome: many genes spread along single DNA molecule

[i.e. all the recipes in one cookbook of the cookbook set]
(Modified from Turnpenny 14_F3.2,3.6 and 3.5)
3. Genome: entire collection of genes for a given organism

[i.e. two different cookbook setseach have a recipe for tamales, but have slightly
different ingredients]
I. Flow of Genetic Information (icontinued)
B. Genetic information flows:
1. Heredity: from parent to progeny (perpetuate the information)
2. Expression: during life of cell (utilize the information)
Rule #1: Central Dogma
Rule #2: Exceptions to every rule

(i.e. retroviruses: RNA>DNA)
C. Processes:
1. Replication: DNA new copies of DNA (entire genome) (Ferrier7 F30.1_p411)
Each daughter cell must get a complete copy.
2. Transcription: DNA *RNA

Only some of the information is needed in a cell at a given time
3. Translation: RNA >Protein
The nucleotide alphabet of RNA (A, C, G, U) is translated to the amino acid
alphabet of 20 amino acids
I. F lo w o f G e n e t ic I n f o r m a t io n ( continued )
D. Genetic code
genetic information encoded by sequence of bases in nucleic acid
problem: how to turn nucleotide sequence into amino acid sequence
'genetic code' refers to polypeptide-encoding regions of nucleic acids
DNA ...-C-C-G-C-C-A-T-G-G-T-G-C-T-C-C-C-A-C-A-T-G-T-A-C-G-C-C-A-A-T-G-A-..
...-G-G-C-G-G-T-A-C-C-A-C-G-A-G-G-G-T-G-T-A-C-A-T-G-C-G-G-T-T-A-C-T-..
RNA ...-C-C-G-C-C-A-U-G-G-U-G-C-U-C-C-C-A-C-A-U-G-U-A-C-G-C-C-A-A-U-G-A...
Protein MET VAL LEU PRO HIS VAL ARG GLN STOP
Attributes of the genetic code:

1. nucleotide, amino acid
sequences are c o - lin e a r First Second position Third
position position
(protein sequence follows along u C A G
the nucleotide sequenceno (51end) (3' end)
skips, jumps) U Phe Ser Cys
Tyr U
2. 'tr ip le t' c o d e Phe Ser Tyr Cys c
Leu Ser Stop Stop A
3 nucleotides > 1 amino acid Leu Ser Stop G
Trp
C o d o n : set of 3 nucleotides c Leu Pro His Arg U
on m R N A that codes for Leu Pro His Arg c
one amino acid Leu Pro Gin Arg A
Leu Pro Gin Arg G
A He Thr Asn Ser U

3. Codons are n o n - o v e r la p p in g : He Thr Asn Ser c
(i.e. first 3 nts.first a.a. He Thr Lys Arg A
next 3 nts. second a.a.) Met Thr Lys Arg G
So no overlap, or gap in between G Val Ala Asp Gly U
Val Ala Asp Gly c
Val Ala Glu Gly A
Val Ala Glu Gly G
4 Code is u n a m b ig u o u s :
Each triplet (codon) specifies
a single amino acidNOT more than 1 amino acid.
(i.e. tJUU always codes for Phe; CAG always codes for Gin)
5. Code is d e g e n e r a te : 64 codons > 20 amino acids + 3 'stop' signals
Thus, 61 codons to code for the 20 amino acids
Result: most amino acids are coded for by more than one codon
(i.e. 2 Phe codons; 6 Arg codons, etc.)
6. Code is u n iv e r s a l (almost): Sam e genetic code is used in all forms of life (with some
exceptions)
Exercise to confirm our understanding:
********************************************************************************
E x e r c is e 1. Identify the amino acid specified by the following codons:
Codon: 5-UAC 5-CCG 5-GUA

Amino Acid:
E x e r c is e 2 . List aU possible codons for the following amino acids:

Methionine (Met) Glycine (Givi Leucine (Leu)
Codon(s):
A d d itio n a l P r a c tic e . Repeat these exercises alone or with a partner.
1) Choose any codon (3 nucleotides), then identify the corresponding amino acid.
2) Choose any amino acid and identify all possible codons that specify that amino acid.
3) Which codons represent the stop signals?

II. DNA Structure
O'
A. Primary structure o=(j>-o-
o=
1. linear polymer of deoxyribonucleotides
(a single strand of DNA)
(modified from Ferrier7_F30.2_p412)
2. phosphodiester bond: formed between 3'-OH of
previous nucleotide to 5'-PC>4 of next nucleotide 0 = ^- 0-
3. polaritya single strand of DNA has a free 5 -P0 4 at one end

and a free 3-OH group at the opposite end.
4. phosphodeoxyribose backbone is constant (structural)

0=fj>-0'
5. base is variable (informational)
B. Secondary structure
1. Features of secondary structure

a. two intertwined helices^double helix of DNA
b. polarity: antiparallel
c. phosphodeoxyribose backbones are external

bases are internal: protected from
environment
d. plane of bases is perpendicular to axis of helix

bases are flat in 3-D structure
e. base pairs: A = T
G=C
a purine is always paired with a pyrimidine to keep
spacing across the strands equal
f. base sequence complementarity

(i.e. if you know the sequence of one strand, you know the
sequence of its complementary strand)
g. about 10 basepairs per helical turn

(S. Triezenberg and J. Wang, MSU)
II. DNA Structure
B. Secondary structure
1. Features of secondary structure (<continued)
h. backbones are not diametrically opposite: major groove, minor groove
2. Stabilizing forces: H-bonds and hydrophobic interactions
a. hydrogen bonds within each base pair
(Ferrier7_F30.4_p413)
b. hydrophobic interactions between adjacent (stacked) base pairs

(1) bases are hydrophobic, thus inside the helix
(2) hydrophobic interactions: major force in holding DNA
structure together

217
II. DNA Structure
B. Secondary structure (continued)
3. Implications of secondary structure:
a. The two strands of double helix can be separated (denatured)
b. Two complementary strands can be rejoined (a.k.a. hybridization, re-annealing, or

renaturation)
Types of duplexes that can be formed:

DNA:DNA duplexes
DNA:RNA duplexes
RNA:RNA duplexes
c. Ability of DNA to be denatured and re-annealed plays an important role in

various molecular diagnostic techniques (i.e. FISH, PCR)
d. Replication model
A: T A: T A: T
G :C G :C G :C
C :G C :G C :G
T :A T :A T :A
T :A T :A T :A
A: T A: T + A: T
C :G C :G C :G
C :G C :G C :G
T :A T :A T :A
G :C G :C G :C

II. DNA Structure (continued)
C. Tertiary structure: chromatin and chromosomes
1. Problem: Each diploid human nucleus contains about 6 feet of DNA. The average
human chromosome contains around 33,000 microns of DNA. A metaphase chromosome is
about 6 microns long and one micron wide. The overall packing ratio of the DNA is,
therefore, on the order of 5,000 to 1.
2. Bacterial nucleoidbacterial DNA is circular and twisted around proteins

(recall no nucleus in bacterial cell)
3. Eukaryotes: Packaged DNA:protein complexes called chromatin. The first

order of packing, and the one that we know the most about, involves the complexing of
the DNA with the histone proteins.
a. Histones
(1) 5 types of histone proteins
H I>wraps linker region of DNA
H2A, H2B, H3, and H4 the 4 core histones
(2) highly conserved
(3) very basic (net charge)so forms electrostatic interactions

with acidic DNA (phosphate backbone is negative charged)
Core of 8 histone molecules: H 1 histone
b. Nucleosomes
(1) 'core' histones: spool
2 of each = 8
(2) DNA: thread

147 bp DNAalmost 2 turns
(3) linker region:

Histone HIwraps the DNA segment (Mesherl3 FS.8'1
b/w one nucleosome and the next
nucleosome
RESULT: appears like beads on a string'
c. Non-histone chromosomal proteins

(1) Stabilize chromatin
(2) Regulate access to the DNA
Note: The complexing of the DNA with the histone

proteins produces an 11 nm fiber of chromatin
(beads-on-a-string chromati n).

II. DNA Structure
C. Tertiary structure: chromatin and chromosomes (continued)
d. Higher-order structures: Solenoid (~30 nm fiber) Chromatin Chromosome
The next order of packing involves the coiling of this fiber of chromatin. The
folding is highly organized and very reproducible. Each chromosome, consisting
of a single molecule of DNA from one end to the other, assumes a characteristic
size and shape each time it condenses during cell division.
unfolded DNA
double helix
molecule
beads-on-a-string'
chromatin
30-nm chromatin
fibril
chromatin fiber
with loops of
chromatin fibril
anchored into
chromosome
scaffold
loosely arranged
chromatin fibers in
euchromatin and
tightly packed
chromatin fibers
in heterochromatin
metaphase
chromosome
b
(Pawlina7_F3.3_p77)
II. DNA Structure
C. Tertiary structure: chromatin and chromosomes (continued)
4. Genome: The total distinct DNA in a cell of a given organism.
a. Concepteach cell contains 1 or 2 copies of its genome

(i) Haploidcell with one copy of each chromosome
Humanshaploid number is 23 (i.e. gametescontain only one member of
each chromosome pair)
(ii) Diploidcell with two copies of each chromosome
Humansdiploid number is 46 (i.e. somatic cells)
(iii) Polyploidabnormal cell with more than two copies of each chromosome
b. The number of chromosomes is species specific
c. Human beings are diploid organisms, with 23 pairs of chromosomes (every somatic cell
contains a set of 46 chromosomes). One member of each chromosome pair is inherited from
the mother while the other member of the pair is inherited form the father. Sex
chromosomes are identical in the female (XX) and different (XY) in the male.
\Metavhor: Cookbook] In the kitchen, there are two sets of cookbooks: one set passed
down from Moms family and another from Dads.
Dad's Mom's
Cookbooks Cookbooks
(R. Ritchie, MSU)
(i) Each set has 23 books where one book = one chromosome.
Each book is themed by country of origin. Book 1 (both Moms set and Dads set)
contains recipes from Afghanistan, etc.
The recipes in the cookbooks from Mom and Dad are for the same dishes but have
slight variations in ingredients. In the book containing Mexican recipes, for example,
Moms family cookbook has beef tamales while the corresponding one from Dads
has pork tamales.
(ii) Each recipe in a book corresponds to a gene.
d. Throughout most of the cell cycle, the DNA is stretched out or attenuated and the
chromosomes themselves are not visible by light microscope. During cell division,
however, the DNA condenses and the 23 pairs of metaphase chromosomes with their
characteristic sizes and shapes become visible.
II. DNA Structure
C. Tertiary structure: chromatin and chromosomes {continued)
5. Eukaryotic chromosome structures

[Remember we see chromosomes only in the process of cell division when the DNA
condenses!]
a. Each pair consists of two homologs:
(i) One homolog was inherited from mother, the other from father.
(ii) Each gene is inherited in two copies, or alleles: one allele derived paternally,
the other maternally
(iii) Exception: Sex chromosomes (vs. Autosomes)
Centromere (DNA)
Kinetochore (protein)
P arm: the short arm (p for petite)
Q arm: the long arm (q is the next
letter in the alphabet)
Sister chromatids
Homologous pair (R. Ritchie, MSU)
b. Telomere:
(i) Tip of each chromosome, capped with 3-20kb of TTAGGG repeat common to all
human chromosome ends (This is the portion of the chromosome implicated in aging
and cell death). Just inside these common sequences is a 100-300kb region of
subtelomeric repeats. These repeats are shared among subsets of human chromosomes.
Just proximal to the subtelomeric repeats is the unique chromosome specific telomeric
DNA which contains the most distal unique sequences (genes) for each chromosome.
There is very high density of genes at the telomeres.
(ii) Maintain the discreteness of chromosomes, preventing them from joining with other
chromosomes or sticking together.
{TTAGGG)* 3-20 kb
RZ3 Tctomcxc Associated Repeats
I I Unique DNA
100-3Q0kb (R. Ritchie, MSU)
II. DNA Structure
C. Tertiary structure: chromatin and chromosomes
5. Eukaryotic chromosome structures (continued)
c. Centromere:
(i) The primary constriction of the chromosome and underlying DNA, seen only when
the chromosomes are condensed. Alpha satellite DNA (171bp tandem repeats, critical
for attachment of chromosome to microtubules of the spindle fiber during cell division)
is one known DNA component of the centromere.
(ii) Interacts with microtubular proteins to act as the center of movement of the
chromosome during mitosis and meiosis.
(iii) Kinetochore refers to the protein structure on either side of the centromere, and
closely associated with it. Many proteins have been identified as playing an important
role in the centromere structure and function.
d. Euchromatin: lightly packed form of chromatin that is rich in gene concentration, it has
less compact structure
(i) DNA is often actively transcribed
(ii) Found in both eukaryotes and prokaryotes
e. Heterochromatin: tightly packed form of chromatin that is poor in gene concentration, it

consists of DNA which is limitedly transcribed, may be differentially condensed at various
stages of the cell cycle. It is not present in prokaryotes. There are two types of
heterochromatin:
(i) Constitutive heterochromatin
DNA that is never transcribed
Contains highly repetitive DNA
(ii) Facultative heterochromatin KwWC r /
Can be genetically active (euchromatin) but is turned
off or becomes genetically inert during some part of i v - "
development of the organism
The late replicating or inactive X in females is an
example
(Pawlina7_F 3.2_p76)
All cells within ah organism have the same DNA
but different chromatin structure, which determines
tissue-specific function.
Kidney and liver are
shown as examples below.
f. Tissue-specific chromatin alterations: differences in the
Kidney chromatin structure (euchromatin and heterochromatin) within
tissues.
(i) stably inherited changes in the chromatin structure
maintained with cell division
(ii) different chromatin structure in different tissues dependent on
Kidney
chromatin the functions carried out by the tissue
Liver
III. GENOMICS: the study of the interaction of sets of genes.
We have talked about the DNA structure. Now we need to know about the information contained
in the DNA of a given cell or organism.
A. Genome Organization:
1. General information:
a. Mitochondria and chloroplasts, which are organelles, have their own DNA content. All
mitochondrial enzymes, though, are not coded for by mitochondrial DNA. Many of the
mitochondrial enzymes are still coded for by nuclear DNA.
b. Viruses can have either DNA or RNA as genomes.
2. How much genetic information is present in human DNA?
a. The total amount of DNA in the human genome is approximately 6.4xl09 base pairs per
diploid cell. Each diploid human nucleus contains about 6 feet of DNA. However, only
about 2% of the human genome encodes proteins.
3. What kinds of sequences are found in human DNA?
a. Genes: These are the sequences that encode proteins (i.e. are transcribed and
translated); the genes that encode functional RNAs (i.e. are only transcribednot
translated); as well as pseudogenes that used to code for proteins evolutionarily, but are no
longer transcribed and translated currently in any cell of the organism.
(i) protein coding genes (i.e. enzymes, structural proteins)
(ii) RNA-coding genes (i.e. rRNA, tRNA, microRNAs, ribozymes)

[Note: also known as nonprotein coding RNAs (ncRNAs)]
(iii) pseudogenes (i.e. inert old genes, but still in genome)
b. Regulatory sequences: These are the sequences that are not transcribed or translated,
but control the transcription of a gene or set of genes. These sequences control where
(meaning which cell) and when the gene is turned on or off in response to various
needs of the cell and/or organism. These are the sequences known as promoters. silencers,
and enhancers.
c. Spacer DNA: These are the sequences that make up the introns (the non-coding
regions of genes) or lie between genes, regulatory sequences, or gene clusters on
chromosomes. There are two major classes of spacer DNAthe unique sequence
spacer DNA and repetitive DNA.
(i) Unique sequence spacer DNA \ These are spacer DNA sequences that typically
appear once in the genome. For example: there might be a particular sequence that
occurs as an intron (or as part of an intron), but this particular sequence of spacer DNA
is not seen elsewhere in the genome either in an intron or as any other type of spacer
DNA.
III. GENOMICS:
A. Genome Organization:
3. What kinds of sequences are found in human DNA?
c. Spacer DNA:
(i) Unique sequence spacer DNA : (continued)
Note that not all introns are totally unique sequence spacer DNA. Repetitive DNA
elements, such as the micro- or minisatellites (discussed below) can be a part of an
intron sequence.
(ii) Repetitive DNA: These are DNA sequences that are repeated many times
throughout the genome, often thousands of times. These repetitive sequences
account for -55% of the genome.
There are TWO types of repetitive DNA:
(a) Dispersed Repetitive DNA: [Accounts for 45% of the genome.] The dispersed
repetitive DNA sequences are also known as transposable elements (a.k.a.
transposons). These are the DNA sequences that can jump into other locations in
the genomeand can cause diseases if they interrupt genes or disrupt how genes are
regulated based on their new location in the genome. These are dispersed
repetitive DNA sequences because each repeat is scattered throughout the genome.
The two most common types of dispersed repetitive sequences are:

SINES (short interspersed elements)
LINES (long interspersed elements)
(b) Satellite (a.k.a. tandem) Repetitive DNA:

[Accounts for 10% of the genome ] These are repetitive DNA sequences that ARE
repeated in tandem (right next to each other). For example a 3 basepair repeat
sequence of GTC is repeated 4 times in order[i.e. GTCGTCGTCGTC], These
sequences are associated with centromeres and telomeres and play a structural role in
the chromosome.
There are 3 types of satellite DNA:

Microsatellite: 1-13 bp repeat unit; blocks of DNA hundreds of bp long
Minisatellite: 14-500 bp repeat unit; blocks of DNA thousands of bp long
a-satellite: always a 171 bp repeat unit; blocks of DNA millions of bp long;
located near centromeres
Clinical Relevance:
The micro- and mini satellites DNA sequences are important because they vary in
length among individuals and are useful in tracking the inheritance of disease-
causing genes in families.
Microsatellite sequences known as trinucleotide repeats are normally present in

certain genes but can undergo expansion (microsatellite instability) leading to
disease (e.g. Fragile X syndrome).
DNA Replication
N ow that w e have some concept o f the general flow o f genetic inform ation and the structure
o f DNA, w e will look m ore closely at the m echanism s underlying those events. W e will first look at
the m echanism o f D N A replication.
Suggested Reading:
Ferrier: Chapter 30: D N A Structure, Replication, and R epair - from Steps in Prokaryotic DNA
replication through Eukaryotic DNA Replication
Turnpenny and Ellard: Chapter 2: The Cellular and M olecular Basis o f Inheritance - DNA: The
Hereditary MateriaD - Replication only (p. 14-15)
Ferrier [available online with the book look under: student resources (if you own the book) or
associated video and audio (if you are using the online M SU library resources)]:
Animation: D N A Synthesis (there are tw o options: O verview or The Details)
https://www.voutube.com/watch?v=dKubvIRiN84
Objectives:
1. Define the process o f D N A replication.

2. Explain the purpose o f D N A replication.
3. Identify w here D N A replication takes place in the cell for both prokaryotes and eukaryotes.
4. Explain the stages o f the D N A replication process in the cell for both prokaryotes and
eukaryotes, (primer, leading strand, lagging strand, origin o f replication, segregation, primase,
D N A polym erase, D N A ligase, topoisom erase, gyrase, helicase, single-stranded D N A -binding
protein (SSB)).
5. Explain how the cell cycle is carried out in the cell (Interphase, Go, Gi, S, G 2 and M phases).
6. D escribe how the cell cycle is regulated (cyclins, Cdks, p53 and pRb) and how disruption o f key
elem ents m ay lead to the developm ent o f cancer.
Measurable Outcomes: What you should be able to do...
1. D escribe the roles o f tem plate and prim er in D N A synthesis. (Obj. 4)

2. Define sem i-conservative replication. (Obj. 1 and Obj. 4)
3. Explain how sem i-discontinuous replication (leading and lagging strands) arises from the 5' to 3'
direction o f polym erization. (Obj. 4)
4. Recognize or draw diagram s representing bi-directional replication from specific origins for
linear and circular genomes. (Obj. 4)
5. Identify the prim ary events in the follow ing stages o f replication: pre-priming,
prim ing/initiation, elongation (leading and lagging strands), term ination, segregation. (Obj. 3
and Obj. 4)
6. Define the roles and attributes o f the follow ing proteins: primase, D N A polymerase, DN A
ligase, topoisom erase, gyrase, helicase, single-stranded D N A -binding protein (SSB). (Obj. 4)
7. D istinguish the activities o f the different types o f D N A polym erase in both eukaryotes and
prokaryotes. (Obj. 1 and Obj. 4)
8. D escribe the fidelity m echanism s o f DN A polym erases, including the role o f 3' to 5' exonuclease
and 5' to 3' exonuclease activities. (Obj. 4)
9. D escribe the problem encountered by lagging strand synthesis at the ends o f linear D N A
molecules. Identify the strategy (including the novel features) by w hich telom erase addresses
that problem. (Obj. 4)
10. Understand the changes that happens to the cell during interphase and these changes correlate
w ith preparing the cell for division. (Obj. 2 and Obj. 5)
11. Identify the prim ary points at w hich the cell cycle is regulated. (Obj. 5 and Obj. 6)
12. Understand how cyclins and cyclin-dependent kinases (Cdks) regulate the cell cycle. (Obj. 5 and
Obj. 6)
13. D escribe the function o f pRb and p53 in the regulation o f the cell cycle. (Obj. 6)
14. Understand the clinical im plications o f disruptions to pRb and p53 and how it may lead to
disease. (Obj. 6)
I. DNA Replication: Flow of Genetic Information to Progeny
A. D N A replication is inherently associated w ith cell division, in tw o ways:
1. D ont replicate D N A unless cell is destined to divide.
2. The cell should not divide until the entire D N A is replicated.
B. A prim ary concern is FIDELITY: the cell m ust preserve the integrity o f genetic
information, and so the D N A replication machinery m ust be very accurate.
II. Six General Features of DNA Replication (for prokaryotes and eukaryotes)
A. R eplication requires a template

1. Always m ake a copy from a tem plate g,
2. N ever de novo (from scratch) ^ Primase

[M ust use the tem plate to dictate the order in
w hich to string together the nucleotides ]
5
5 3
RNA
B. Replication enzymes require a primer ^ DNA polymerase
1. DNA polymerases require a short 3'

nucleotide sequence already in place 5
new DNA
2. DNA polymerases always m ust have a free 3 -OH group available (S. Triezenberg and
J. Wang, MSU)
for attachment o f the incom ing nucleotide
3. Prim er is R N A segm ent (usually) [RNA polym erases do N O T require a primer]
C. R eplication is semi-conservative
1. Each parent strand is a template
2. Each parent strand is conserved

(not degraded)
3. The tw o parent strands do N O T stay

together
4. Thus is called semi-conservative

replication
One parental strand is conserved

(Ferrier7_F 30 .8_p415 ) in each of the two new double helices
II. Six General Features of DNA Replication (
D. DNA chains are m ade in the 5' 3' direction always

(N ote: RNA also synthesized 5' 3' direction always)
DNA template
3
5
new DNA
1. Problem : H ow to replicate both strands?
(Because tem plate A LW AYS read 3 to 5 so

that new strand is ALW AYS made 5 to 3 )
\ Replication fork
2. Solution: "semi-discontinuous replication"

a. One strand is continuous (the leading strand)
b. Other strand is discontinuous m eaning

synthesized in short pieces called
Okazaki fragments (the lagging strand).
These strands will ultim ately be joined
together to form one continuous strand o f
DNA.
Lagging strand Leading strand

Replication bubble
Origin of replication
3 ^ 5 'U l i b j
^ Replication forks "

Okazaki fragments j Leading strand Lagging strand
(F errier7_F30.14_p418)
II. Six General Features of DNA Replication (continued)
E. Replication begins at specific start sites (origins)termed origin of replication
single replication origin multiple replication origins
l l i l l
Bacterial chromosome Eukaryotic chromosomes

Have multiple replication origins because:
1. Have many chromosomes
2. Each chromosome is huge and tightly packed
3. Multiple replication origins per chromosome allow
for faster replication of each chromosome.
F. Replication (usually) proceeds bi-directionally from an origin

[Note: The arrows in the figures below do NOT indicate leading and lagging strand
synthesis. The arrows only indicate the bi-directionality of replication of the replication
bubbles.]
Prokaryote Eukaryote
III. DNA Replication process E. c o li (prokaryotic)
(Illustrates the 6 features; simpler; know more details; target of many antibiotics)
A. What? DNA replication (semi-conservative)results in new duplex DNA molecules in

which one strand is newly synthesized and paired with its template strand (parent strand).
B. Why? To maintain (preserve) entire copies of the organisms genome to be passed down to
its progeny (daughter cells).
C. Where? In eukaryotesthe nucleus of cells

In prokaryotesthe cytoplasm (b/c no nucleus)
D. How? Overview: stages of DNA replication process
b. Priming /
e. Segregation

III. DNA Replication process E. c o li (<continued)
E. Details of stages in E. coli DNA replication

1. Pre-priming Stage: Events
a. origin recognition (oriC)
Involves several proteins & enzymes that:
recognize origin and start strand separation
template preparationhelicases unwind
SSB (single-stranded DNA-binding protein)
prevents re-annealing of strands,
binds to ssDNA strands
GULATED STEP (S. Triezenbergand J. Wang, MSU)

If this pre-priming process starts, replication MUST be carried out completely
2. Priming/Initiation Stage
a. Primasemakes an RNA primer (-10-12 ribonucleotides long) anywhere near an

origin of replication
(1) Primase is an RNA polymerase, required for DNA synthesis

[RNA polymerases do NOT need to have a free 3-OH for attachment]
(2) No proofreading of RNA primer
(3) RNA primers must be removed later (from both leading and lagging strands)
!h
6 6 6 '6 6 6 6 iV 7
b. NOTE: DNA polymerases which catalyze attachment of deoxynucleotides for synthesis

of DNA during elongation stage ALWAYS requires a primerneeds free 3-OH end.
III. DNA Replication process E. coli
E. Details of stages in E. co li DNA replication (<continued)
3. Elongation Stage
a. Leading strand:
(1) Continuous5>3
also have RNA primers at the beginning of strands, which will be removed
(as discussed below with lagging strandssee DNA pol. I and DNA ligase)
(2) DNA polymerase HI: (two enzymes in one protein)
5 to 3 DNA synthesis (DNA polymerase enzyme)
makes the DNA polymer by extending from the 3-OH of the RNA primer
[Recall DNA template is being read 3 to 5]
3 to 5 exonucleasethe proofreading function; removes an incorrectly
incorporated nucleotide from the NEWLY synthesized strand at the free 3-OH end.
[NOTE: exonucleases remove nucleotides from the ENDS of DNA]
b. Lagging strand:
(1) Discontinuous
Small fragments synthesized in 5 >3 direction
(2) Primase
Each fragment has an RNA primer
Primed often, about every 100 nts
(3) DNA polymerase III (same enzyme as listed above for leading strand)
Again, synthesizes DNA by extension from the RNA primers
(4) DNA polymerase I: (THREE enzymes in one protein)
5 to 3 DNA synthesis (DNA polymerase enzyme)
3 to 5 exonuclease (proofreading functionlike DNA pol. Ill)
removes mismatched nucleotides in 3 > 5 direction
5 to 3 exonuclease (snowplow function)
removes the RNA primers in the 5 >3 direction (on both strands)
(5) DNA ligaseseals nicks between 2 fragments (on both strands)
[Where the RNA primers were removed and replaced with deoxynucleotides]
c. Replication fork:
[See figure belowwhich shows ONE replication fork of a replication bubble]
(1) helicase: separates parent strands
(2) SSB (single-stranded DNA-binding protein): prevents re-annealing of strands
(3) gyrase: a topoisomerase that unwinds in front of the fork
Relieves the overwinding by putting in negative unwinding
gyrase is a great drug target (i.e. ciprofloxacin acts on gyrase)
III. E. 3. Elongation Stage (continued)
d. Details of lagging strand synthesis
(1) DNA polymerase ///synthesizes the new strand until it reaches an RNA primer
(2) DNA polymerase I removes the RNA nucleotides starting at the free 5 -P0 4 end (5
to 3 exonuclease) one at a time, and attaches the correct deoxynucleotide (5 to 3
polymerase activity). The 3 to 5 exonuclease then proofreads the new
deoxynucleotide to be sure it is correctand if not removes it.
(3) DNA ligase seals the nick between the 2 fragments
new RNA
first RNA primer
primer
5'
5' template 3'
DNA pol III
5'
DNA pol III
DNA pol I : excise RNA primer, extend DNA

5' 3'
DNA ligase
3'
e. Global view of replication forks (replication bubble)
4. Termination Stageoccurs when the two replication forks meet (recall circular DNA)
5. Segregationlinked circles are separated using topoisomerases (enzymes that nick and
break either 1 or both strands of DNA). In this case both strands are clipped to separate the two
circular pieces of DNA.
III. F. Regulation of DNA synthesis for E.
1. Time for entire cycle (initiation to cell division)about 60 minutes for E.

2. Interval between successive cell division: doubling time is about 20 minutes
(can start a second round of replication before the first round is done)
IV. DNA Replication process Eukaryotes

A. Complexities
1. Packaging
a. DNA is packaged as chromatin
b. Must unwind the DNA from the histones (so to separate parent strands takes more time)
c. Must re-package daughter copiesactually overpackage (highly condense)
as chromosomes, to keep from tangling. (So now can see individual chromosomes)
2. Multiple origins
a. Multiple origins on each chromosome
b. Poses regulation problem
(1) Initiate each origin ONCE
during each cell cycle
(2) CANNOT replicate DNA

multiple times per cycle
like E. coli
Newly synthesized DNA
3. Eukaryotic DNA polymerases: (S. Triezenberg and J. Wang, MSU)
a. priming/initiating enzyme:
(1) DNA polymerase a (alpha): part of protein complex that initiates DNA synthesis
(2) Has primase activity puts in the RNA primer (on both leading strand and Okazaki
fragments of lagging strand)
(3) AND has DNA polymerase activity puts in SHORT segment of DNA
still must see that free 3OH group
does not go far because it does not have the proofreading function
POLY PROOF
b. m ajor replicative enzymes: MERASE
FUNCTION
READING
(1) DNA polymerase c (epsilon) DNA synthesis on
the leading strand Pot a * Contains p r im a s e
(2) DNA polymerase 5 (delta) elongates the Okazaki (alpha) Initiates DNA
fragments o f the lagging strands synthesis
(3) Highly processive the m ajor DNA synthesis P O lfi * Repair
enzymes (beta)
Each DOES have the 3 to 5 proofreading exonuclease P o tS * Elongates Okazaki +
activity (delta) fragments of the
tagging strand
c. RNA prim ers from leading and lagging strands are Pah: Elongates the +
rem oved by RNase H and flap endonuclease-1 (FEN1) (epsilon) leading strand
N O T DNA polym erase P o ly * Replicates +
[Different from DNA polym erase I in E. coli replication] (gamma) mitochondrial DNA
d. DNA polymerase (3 (beta) involved in gap filling in DNA repair (Ferrier7_F30.23_p423)
e. DNA polymerase y (gamma) replicates mitochondrial DNA

A. Com plexities ( continu
4. Linear DNA W hat prim es the synthesis at the ends o f chrom osom es?
a. The problem: oriain
(1) CANNOT prim e last bit o f DNA at 3 Leading strand 5
3 -end o f tem plate
----
(2) Because NO free 3 -OH 5 3
(3) RN A prim er can start at very end, but Lagging strand

after it is removed, there is no way for a DNA
polym erase to fill it in because no free 3 -OH
b. The structure: telomeres = the repeating sequence at ends o f chrom osom es

3
^ 5
... AGGGTTAGGGTTAGGGTTAGGGTT-3
... TCCCAATCCCAA-5
c. The solution: telomerase (a.k.a. telom ere synthetase)
(1) strategy: extend the 3 ends o f the PA RENT STRANDS
How? Because always need a tem plate
3
5
(2) m echanism: telom erase has a

portable R N A tem plate
reverse transcriptase activity

(i.e. RN A tem plate DNA synthesis;
w hich contradicts central dogma)
Note: still cannot replicate very ends o f strands,

but OK now because is ju st a repeat sequence (i.e. does
N O T contain a code for a gene)
236
IV . DNA R ep licatio n process E u k a ry o tes
A. Complexities
4. Linear D N A W hat prim es the synthesis at the ends o f chrom osom es? (continued)
d. Implications:
(1) Cell aging telom erase is m ost active in germ cells
(as a cell ages, telom erase activity decreases, cell dies)
(2) C ancer cells telom erase is always active, therefore cells do not die
B. Cell cycle: coordination o f events surrounding eukaryotic D N A replication and cell division
(mitosis).
Prophase
( 1 h)
Metaphase
(<1h)
Anaphase
(< 1/2 h)
Telophase
(minutes)
Source: Mescher AL: Junqueira's Basic Histology, 13th Edition: www.accessroedlclne.com (Vleschcrlt 1' 3 12)
Copyright cc The McGraw-Hill Companies, Inc. All rights reserved.
B. Cell cycle: {continued)
1. Interphase: period between successive mitosis. A typical cell spends most of its life in
interphase doing whatever it is programmed to do. (Go for non-dividing cells, indefinitely in
Gi. Go is a modified Gi phase.) There is then a period of DNA synthesis (S) in which
chromosome duplication occurs followed by a further gap (G2) in time waiting for mitosis to
begin. During this gap (G2), essential proteins and cofactors are produced necessary for mitosis
to occur.
a. Gi (Gap 1) phase: interval between mitosis and DNA replication.
Cell carrying on its activities, preparing to duplicate its DNA content
Cell grows in size and accumulates nutrients
Cells accumulate the enzymes and nucleotides required for DNA replication
b. S (synthesis) phase: DNA replication occurs.
The chromatid of each chromosome is replicated - 2 chromatids per chromosome
(chromosome X-shaped)
c. G2 (Gap 2) phase: interval between S phase and next mitosis.
Some DNA repair occurs
Chromosomes begin to condense in preparation for mitosis
Proteins required for mitosis accumulate
Short period in preparation for mitosis
d. Go phase: Cells that have stopped dividing and left the cell cycle. These quiescent cells
can reenter the Gi phase to resume cell division.
Quiescent cells - fibroblasts, liver cells, smooth muscle cells, and endothelial cells
(may reenter cell cycle under certain conditions)
Terminally differentiated - skeletal muscle and neurons (cannot reenter the cell cycle)
2. M phase: Includes all the phases of mitosis.
1 chromosome 1 chromosome
(after replication) (after mitotic division)
(Tumpennyl4_F 3.1)
IV. DNA Replication process Eukaryotes (continued)
C. Regulation of cell cycle: accomplished by the presence of checkpoints at critical points in the
cell cycle. During the checkpoints the completion of critical events is monitored and, if necessary,
progression to the next stage of the cell cycle is delayed.
1. Gi checkpoints: "START"
a. Decide to go/not go into DNA Replication
b. Gi DNA-damage checkpoint:
Is DNA OKneed to repair first?
c. Restriction point (R point):

Activities occurring before this point depend on extracellular resources such as
mitogens and growth factors. Once the R point is passed, the cell cycle no longer
depends on these extracellular resources.
Considerations: Are cells nutrients, size, and environment favorable?
d. This checkpoint is mediated by the interactions between retinoblastoma susceptibility

protein (pRb) and essential transcription factors (E2F).
Hyperphosphorylated pRb releases E2F - E2F is a transcription factor that activates
genes needed for cell cycle progression.
[growth factor stimulation signaling via MAP/PI3 kinase phosphorylation and
activation of cyclinD/CDK4/6 complex -> pRb phosphorylation]
Hypophosphorylated pRb binds to E2F - turns off genes needed for cell cycle
progression (blocks cell cycle progression).
(Chandarl_F21.5)
Clinical Relevance: Hereditary retinoblastoma is an inherited eye malignancy caused

by a mutation in the pRb gene. The mutant pRb protein is unable to stop the cell cycle in
Gi allowing unregulated progression through the remainder of the cell cycle.
C. Regulation of cell cycle:
DNA
1. Gi checkpoints: "START" (continued) da matte
e. p53:
Is phosphorylated (activated) when there is DNA
damage.
Activation induces transcription of p21 If damage cannot

be repaired,
(inhibitory protein) - stops cell cycle progression. programmed cell
death occurs,
Cell tries to repair DNA damage. If damage not
repaired -> programmed cell death (apoptosis). If damage can be
repaired, the cdk
inhibitor, p21,
Clinical Relevance: More than 50% of human stops the cell
tumors contain a mutation or deletion of the p53 cycle,
gene and those who inherit only one functional
copy of the p53 gene will most likely develop

tumors in early adulthood. Li-Fraumeni syndrome Upon DNA
repair. DNA V -
occurs when a mutation in p53 is inherited and is replication
characterized by multiple types of tumors. Some can proceed
tumors with p53 mutation are resistant to and the active
phases ot the
radiotherapy because apoptosis will not be cell cycle
triggered in the absence of functional p53. can be
re-entered.
f. Tumor Suppressor Genes:

encode proteins that form a network of
checkpoints that help control cell division and
suppress inappropriate cell proliferation.
(Chandarl F21.6)
the product of these genes apply brakes to cell proliferation and may function as
transcription factors, cell cycle inhibitors, signaling molecules, cell surface receptors and
regulators of cellular responses to DNA damage.
Clinical Relevance: Human Increased

papillomavirus (HPV) strains TERT lelomerase
16 and 18 are associated with expression
cervical cancer. In cervical

cells infected with these viral p53 Inhibition of p 53
strains of HPV: N. C
- Viral protein E6 - binds Immortalization
to p53 and targets it for Increased cell
degradation via the proliferation
proteasome pathway. Genomic instability
- Viral protein E7 - binds
to pRb and prevents it
from inhibiting
proliferation.
- Both p53 and pRb are
inactivated leading to
possible upregulated cell
cycle progression and
malignancy.
C. Regulation of cell cycle: (continued) tW
2. S DNA-damage checkpoint:
a. Monitors the quality of replicating
DNA. [ Cell in Gi j
b. Slows the rate of DNA synthesis if

DNA damage occurs during S phase.
c. BRCA1 plays a role in the repair of
double-strand DNA breaks.
3. G2 checkpoint:
a. Is replication complete?
b. Is the cell ready to divide?
4. Metaphase checkpoints:
a. Spindle-assembly checkpoint:
Are all chromosomes attached to the mitotic spindle? (Chandarl _F21.8)
If OK, cells will progress into anaphase.
b. Chromosome-segregation checkpoint:
Have all the chromosomes been correctly separated?
c. Now cells divide if everything is OK.
spindle-assembly chromosome-segregation
checkpoint checkpoint
G2
checkpoint
G! DNA-damage
checkpoint
S DNA-damage
checkpoint
restriction
checkpoint
(Pawlina7_F3.10_p84)
IV. DNA Replication process Eukaryotes (icontinued)
D. How is the cell cycle regulated?
1. Two key classes of proteins control the progress of a cell

through the cell cycle: cyclins and cyclin-dependent
kinases (Cdks).
a. Different cyclins are present during different phases of
the cell cycle.
b. Each cyclin activates one or more specific Cdk. When
cyclins are paired with their appropriate Cdk, the Cdk
becomes phosphorylated leading to activation of its
kinase activity and the cell cycle is permitted to
progress.
c. To control cell cycle progression, the amount and type
of cyclins varies.
Note: the Cdk concentration in the cell does not
change.
d. When a cyclin-Cdk complex is formed, it activates the
kinase activity of the Cdk.
e. Each activated Cdk phosphorylates and activates specific proteins, transcription factors
for specific sets of genes, and cytoskeletal subunits - triggering the activities needed for
phase specific functions.
f. When the set of activities of that phase are completed, the cyclin controlling that cell
cycle phase is removed by ubiquitin-mediated proteasome degradation.
g. The cyclin that promotes activities for the next phase will take over.
Note: cyclin A pairs with both CDK2 and CDK1 to regulate different portions of the
cell cycle.
Targets: nuclear laminins, hitorte H1,

chromatin-associated proteins, and
centrosomal proteins.
Targets specific
proteases and
cyclin B.
Cyclin A-Cdkl
Targets:
Targets DNA phosphorylates pRb -
polymerase and other release E2F
proteins for DNA
replication.
Further activates E2F-mediated gene

transcription and phosphorylates p53.
(Modified from: Pawlina7_F3.1 l_p86) 242
Chromosomes during Mitosis and Meiosis
Our gametes (sperm or egg) contains a haploid content o f our genome (23
chromosomes) that we will pass onto our offspring, (1 haploid +1 haploid = a diploid).
These 23 chromosomes contain a new mixture o f gene content, derived from each
parent, thus encompassing a novel gene set to contribute to the diploid content o f the
next generation.
Obviously, aberrant inheritance o f too much or too little chromosomal material will
have serious consequences.
Understanding the dynamics of normal chromosomal biology will facilitate
understanding of the etiology and ramifications of chromosomal diseases.
We will focus on the process o f a single somatic cell becoming two new daughter
cells, while preserving the amount o f DNA that should be contained within a cell, a
process called MITOSIS. In contrast to this process, in the case o f a germ cell (sperm
or egg), cell division is more complicated as a reduction o f genetic content to Vi of
original content is required for each gamete generated. This process (MEIOSIS), will
generate female or male gametes, depending on the sex o f the individual.
Suggested Reading:
Turnpenny and Ellard: Chapter 1: The History and Impact o f Genetics in Medicine -
Gregor Mendel and the Laws o f Inheritance (p. 3-5); Chapter 3: Chromosomes and
Cell Division - from Cell Division through Gametogenesis (p. 38-42)

https://www.youtube.com/watch?v=zfEKlTBJsrO [Mitosis Animation]
https://www.youtube.com/watch?v=rqPMpOUOHOA [Meiosis Animation]
https://www.voutube.com/watch?v=zGVBAHAsiJM [Mitosis, meiosis, cross-over

(recombination) and independent assortment]
http://www.pbs.org/wgbh/nova/bodv/how-cells-divide.html [Comparison - mitosis vs

meiosis]
Objectives:
1. Understand the stages o f mitosis how they correlate with the transmission o f genetic
material in somatic cells.
2. Understand the stages o f meiosis and how they correlate with the transmission of
genetic material to germ cells.
3. Describe the principal features and genetic implications o f homologous
recombination, including the significance o f chiasmata structure and the pachytene
stage o f meiosis I.
4. Explain the stages of gametogenesis for both male and female, including the
significance o f dictyotene stage of meiosis I in oogenesis..

1. List in correct sequence the stages o f mitosis (somatic cell division) and the
chromosomal events that occur in each stage (focus on the bold and/or italicized
content). (Obj. 1)
2. List in correct sequence the stages o f meiosis (germ cell division) and the
chromosomal events that occur in each stage (focus on the bold and/or italicized
content). (Obj. 2 and Obj. 3)
3. Compare and contrast the stages o f mitosis and meiosis, distinguishing the
separation o f homologous chromosomes versus the separation o f sister chromatids
(Obj. 1 and Obj. 2)
4. Correlate chromosomal events in meiosis with the Mendelian concepts of
independent assortment and segregation o f alleles. (Obj. 2 and Obj. 3)
5. Define the terms alleles, co-segregation, and recombination. (Obj. 3)
6. Describe the phenomenon o f crossing-over (genetic recombination), segregation
and independent assortment; explain how recombination affects segregation of
alleles. (Obj. 2 and Obj. 3)
7. Identify haploid and diploid stages o f gametogenesis. (Obj. 4)
8. Describe each stage of gametogenesis with respect to mitosis and meiosis. (Obj. 4)
There are two types of cell division:
Mitosis:
Occurs during cell division o f somatic cells (somatic cell division)
A single cell division, one cell becomes two: the cell begins mitosis with 46
chromosomes, each consisting o f 2 chromatids. At the end o f mitosis, there are
2 daughter cells, each containing 46 chromosomes, each chromosome
composed of 1 chromatid
A diploid cell generates an identical diploid cell
There is no change in chromosome number, and
The daughter cells are genetically identical, normally, no recombination takes
place
Meiosis:
Occurs during gamete formation
A diploid progenitor cell generates four haploid gametes
Meiotic recombination (cross over) is frequent
I. Mitosis
Human beings contain trillions of individual cells. As few cells last for an individual
entire lifetime, new ones must be generated to replace those that die, therefore, at
some point during their life, cells will divide.
Diploid cells will generally maintain their diploid DNA complement.
Mitotic cell cycle: alternation of interphase and mitosis
Interphase: betw een divisions.

Gi p h a se (Gap 1): interval betw een m itosis a n d replication (c=DNA content;
n= haploid chrom osom e number).
S p h a s e (synthesis): C ell replication occurs , duplicates its DNA.
G2p h a se (gap 2): interval betw een the S p h a s e a n d n ext m itosis. C ell contains
2 identical copies o f each o f the 46 chrom osom es. These identical chrom atids
p e r chrom osom e are referred to as sister chrom atids. S ister chrom atids often
exchange m aterial du rin g or after the S phase.
[N ote: sin ce the sister chrom atids are identical, th is typically does N O T resu lt
in recom bination]
M phase: mitosis (cell division); for diploid to diploid division, which occurs in
somatic cells, this is referred to as mitosis. Content returns to 46 chromosomes (each
with one chromatid).
Prophase: chrom osom es condense; centrosom es (with duplicated centrioles)
separate an d m igrate to opposite p o le s o f th e cell; m icrotubules radiatin g
fr o m the centrioles begin to fo r m th e m itotic spindle. This stage is heralded by
the initiation of chromosomal condensation. As condensation of the chromatin
occurs throughout prophase, highly condensed regions as well as less
condensed regions develop which begin to correlate with light and dark banding
patterns observed on light microscopy following staining of the chromosomes
with Giemsa or Wright stain. E ach chrom osom e n ow contains tw o stran ds o f
dsD N A (sister chrom atids) which lie p a ra lle l to on e another a n d are
con n ected together a t on e sp o t by th e centrom ere.
(Tumpennyl4_F3.13_p38)
Prometaphase: nuclear m em brane disappears; chrom osom es attach to
spin dle m icrotubules a t their kynetochores. Prometaphase starts abruptly with
disruption of the nuclear envelope, which breaks into
membrane vesicles. The spindle microtubules can now
enter the nuclear region. Kinetochores, which are
specialized protein complexes, mature on each
chromosomes centromere and attach to some of the
spindle microtubules which are then called
kinetochore microtubules. The remaining
microtubules in the spindle are called polar
microtubules and those outside are called astral
(Pawlina7 F3.13_p88)
microtubules.
Metaphase: C hrom osom es are fu lly condensed , line up sin gle f i l e alon g the
(m e ta p h a se p la te , each chrom osom e is attached to centriole by a m icrotubule
fo rm in g m atu re spindle; spin dle fib e r s begin to contract. In metaphase, the
chromosomes become maximally condensed. They become associated with
kinetochore microtubules of the spindle apparatus which align them at the
equatorial plate midway between the two poles of the cell. Polar microtubules
begin to associate in preparation for anaphase. At this phase the chromosomes
are easily visible on light microscopy.
Meiaphase
(T umpenny 14_F3.13_p3 8 )
(M escherl3_F3.14)
Anaphase: C entrom eres divide into two, spin dles p u ll sister chrom atids
tow ard opposite sides o f the cell (centrom ere first) dictated by w h ere the
centrioles form ed. In anaphase, the centromeres (kinetochores) abruptly
separate and each sister chromatid (now referred to as individual chromosomes)
is pulled to its opposite pole by the kinetochore microtubules.
Anaphase
(Mescherl3 F3.14)
Telophase: two nuclear m em branes f o r m , spin dle fib e r s disappear,
chrom osom es decondense an d return to interphase. Once the two sets of
chromosomes arrive at the cell poles, the spindle apparatus begins to dissociate.
The polar microtubules elongate even more, further separating the two poles
within the cell. A nuclear envelope begins to form around the aggregated
chromosomes present at each pole. The chromosomes begin to decondense
within their new nuclei. At this point, mitosis is at an end and the cell enters a
phase of Cytokinesis (cell division).
Telophase
(Turnpenny 14_F3.13_p38)
Cytokinesis (form tw o identical daughter cells): occurs after nuclear division a n d

results in a roughly equ al division o f th e cytoplasm into tw o parts. This occurs by a
process known as cleavage which actually begins
during the anaphase stage. A cleavage furrow
develops as the cell membrane around the mid
section of the cell is drawn inward. This furrow
continues to deepen until it encounters remnants of
the spindle apparatus to form a small bridge-like
structure called the mid-body between the two
nuclei. Eventually, this bridge structure breaks down
resulting in separation of the now independent
daughter cells. (Mescherl3 F3.14)
R etu rn to G1 o f interphase: tw o diploid daughter cells , gen etically identical to the

original cell.
The length of the cell cycle varies considerably from one cell type to another. The
large difference is the length of interphase.
Epithelial cells: ~10 hours

Liver cells: one year
Skeletal muscles and neuron: lose their ability to divide and replicate in adults
When cells stop dividin g f o r a lon g p erio d , they are often sa id to be in the G0phase.
The stages o f mitosis. Two identical cells are form ed from one original diploid cell.
Centriolcs
interphase
Nucledous
Nuclear membrane
Bipolar spindle fiber
Centromere
Turnpenny; Emery's Elements of Mediical Genetics, I4e

Copyright 2011 by Churchill Livingstone,, an imprint of Elsevier Ltd* All rights reserved.
(Turnpenny 14F3.13_p38)
II. Meiosis
Goal: We need to pass on V2 of our genetic information to our offspring. We thus
generate haploid gametes (containing one homolog of each pair, i.e. 23 different
chromosomes)
Meiosis is a specialized form of cell division, a process by which haploid gametes are
formed from diploid precursor cells, takes place exclusively in the gonads, occur only
during gametogenesis.
It takes place in two steps, requires 2 types of cell division:
Step One (meiosis I): Reductive division. reduces 46 chromosomes to 23. 2

haploid cells (23 chromosomes) are formed from a diploid cell (46 chromosomes).
These diploid cells are called oogonia in females and the spermatogonia in males.
Step Two (meiosis II): Equational division, separates the sister chromatids. It is
essentially identical to mitosis in somatic cell, except only 23 chromosomes are
present.
Result: One diploid cell generates four haploid cells, 4 sperms in male; one egg and 2-
3 polar bodies in the females, 3 polar bodies if the second polar body divides. Each
chromosome composed of 1 chromatid.
Meiosis I: It starts with 46 chromosomes and 92 chromatids and has 5 stages:

interphase, prophase, metaphase, anaphase, and telophase, but the prophase in meiosis
I is much more complex and may span (in the oocyte) for 40 years or more.
Interphase I: replication o f chromosomal DNA occurs (similar to mitotic

interphase)
Prophase I: chromatin strands condense, the homologous chromosomes pair

up, side by side, lying together in perfect alignment during a process called
synapsis, this pairing of homologous chromosomes is an important part of the cell
cycle. Chiasmata (chiasma) forms, a cross-shaped structure where the
homologous chromosomes attached form ing a bivalent (see figure on next
page). A bivalent indicates 2 homologous chromosomes in the unit (or a tetrad
o f 4 chromatids in the unit). Each chiasmata indicates a point at which the
homologues exchange genetic material between non-sister chromatids . a
process called crossing over or recombination , it greatly increases the
possible combinations of genes in each gamete and thereby increase the number of
possible combinations of human trait. Chiasmata are positions where
chromosomes touch and crossing over has occurred between chromatids of
homologous chromosomes. The crossing over phenomenon between paternal and
maternal chromatids creates endless possibilities of recombination and therefore
genetic diversity.
5 stages are defined for prophase I:

-leptotene: chromosomes begin to condense
-zygotene: homolog pairs, telomere first and zip down, synaptonemal
complexes form
-pachytene: crossin g over occurs (aka. recom bination)
-diplotene: hom ologs separate, rem ain attached a t chiasm ata
-diakinesis: separation of homologs
In fem a le, there is a special step o f diplotene, called dictyotene, arrested a t 9 h

m onth o f gestation f o r 10-50 y e a rs until ovalution.
E-i'ty' pachytene Late pachytene
Diplotene'
unmans Duikinutiis
Fropnase I
(Turnpenny14_F3.15_p40)
C hiasm ata . The process of chiasma formation and crossing over results in the
exchange of genetic material between the non-sister chrom atids of homologous
chromosomes.
centromeres
chiasma
i
sister chromatids
(http://www.histology.leeds.ac.uk/cell/meiosis.php)
Metaphase I: com plete fo rm a tio n o f spin dle a n d two
centrom eres o f each bivalen t lie on the equatorial plane.
Thus , the chrom osom es line up in pairs along the
m etaphase plate.
MtlAphtM <
Anaphase I: chiasm ata disappear a n d the h om ologous chrom osom es are p u lle d
by the spin dle fib e r s tow ard opposite p o le s o f the
cell. Note: the centromeres do not duplicate and
divide, only half of the original number of
chromosomes migrates toward each pole, consisting
of only one member of each pair of autosomes and
one of the sex chromosomes.
Telophase I: it begins w hen the chrom osom es reach opposite sides o f th e cell.
N ew nuclear m em brane begins to fo rm . E ach o f the tw o daughter cells contain
the haploid num ber o f chrom osom es a n d each chrom osom e has tw o sister
chrom atids. C ytokinesis also occurs in human, i.e. the cytoplasm is divided
approximately equally between the 2 daughter cells in the gametes formed in
males, but in females, nearly all of the cytoplasm goes into one daughter cell,
which later form the egg; the other daughter cell becomes a polar body, a small,
nonfunctional cell that eventually
degenerates.
Meiosis II: similar to mitosis
Interphase II: very b r ie f p h a se, no replication o f D N A occurs, differen t fr o m

interphase I a n d m itotic interphase.
Prophase II: the cell contains only the h aploid num ber o f chrom osom es,
chrom osom es condense a n d the nuclear m em brane disappear, n ew spin dle
fib e r s are form ed.
Metaphase II: spin dle fib e r s p u ll th e chrom osom es into align m ent sin gle f i l e }}
a t th e equ atorial plane.
Metaphase u
Anaphase II: centrom ere o f sister chrom atids sp lit a n d each carries a sin gle
ch rom atid tow ard a p o le o f th e c e ll N ew ly separated sister chrom atids m ay n ot
be identical due to crossin g over.
A ns phase ll
Telophase II: it begins w hen th e chrom osom es reach opposite p o le s o f th e cell.

N ew nuclear m em branes are fo r m e d arou n d each grou p o f chrom osom es. A n d
cytokinesis occurs.
Telophase II
In m ales, the cytoplasm is equally divided into 2 gam etes, th e e n d p ro d u ct

o f m ale m eiosis is fo u r fu n c tio n a l daughter cells.
In fem ales . th e en d p ro d u cts are on e fu n c tio n a l dau ghter cell, 2-3 p o la r

bodies.
The stages o f meiosis. Haploid gam etes are form ed from a diploid cell.
Early pachytene Late pachytene
Zygotono Diplolane
Levtolene Dibikinesis
H d a phase I
Anaphase 1
Telophase I
Metaphase u
A n a p h a s e II
Telophase II
Turnpenny: Emery's Elements of Medical Genetics, I4e

Copyright 2011 by Churchill Livingstone, an imprint of Elsevier Ltd. All rights reserved,
The following table and figure compares and contrasts the major features of
mitosis and meiosis
M itosis M eiosis
Occurs in somatic cells Occurs in germ line cells (gonad)
Takes about an hour Fem ale begins at 3-4 m onths gestation, 1st
division is complete at ovulation, 2nd division is
complete at fertilization.
M ale begins at puberty, com plete in 60-65 days
Chrom osom es do not pair Hom ologous chrom osom es pair (prophase I)*
U sually no chiasm ata or crossing over Chiasm ata and crossing over always occur
1 cell division 2 cell divisions produce 4 daughter cells
N o change in chrom osom e num ber Chrom osom e num ber reduced to one o f each pair
(haploid set in each daughter cell)
N o change in gene content All daughter cells m ay be genetically different,
due to segregation o f chrom osom e pairs and
crossing over betw een homologues
PREMITOTIC/MEIOTIC EVENTS
MITOSIS prophase I MEIOSIS

I I I . M e n d e l s L a w s o f In h e r it a n c e (m eiosis is the basis)
A. Segregation (2nd law):

1. The 2 members of a single gene pair (alleles) are never found in the same
gamete; they always segregate and pass to different gametes.
2. During meiosis, the two chromosomes of a homologous pair will separate
(homologs separate).
(J. Simmons, MSU)
B. Independent assortment (3 rd law):

1. Members of different gene pair assort to the gametes independently of one
another.
2. The segregation of one pair of homologs is independent of and does not affect
the segregation of other pairs of homologs.
3. 223combinations are generated by independent assortment.
(J. Simmons, MSU)

The physical basis of segregation and independent assortment is found in meiotic
anaphase I.
IV. Expanding Genetic Diversity: Recombination

A B C D E F
A. Each chromosome contains many ------
genes a b
1. syntenic genes - genes on the same
chromosome
B
i.e. A, B, C, D in figure at =
right are synthenic genes
B
2. alleles - different forms a gene =
may have in a population (S. Triezenberg and J. Wang, MSU)
(Note: diploid organisms often have different alleles of a gene)
i.e. A and a are alleles in figure
3. co-segregation - syntenic alleles tend to stay together through generations

4. recombination - occasionally co-segregation is disrupted resulting in a new
combination of alleles on a chromosome (see figure above, bottom diagram)
B. Homologous recombination
1. Exchange of DNA fragments between homologous chromosomes
(i.e. between the two copies chromosome #10) A B C D
2. Occurs during meiosis: crossover events a b C d

a. When making gametesegg and sperm cells
b. Homologous chromosomes align A B c d
(i.e. sequence similarity matters)
c. dsDNA strands break and rejoin a D
* Ir
3. Consequence: additional genetic diversity
a. Resulting chromosomes have new combination of
A B c d
alleles
b. Allows for experimentationMAY result in a b c D
increased fitness of the organism (i.e. evolution)
4. Frequency of recombination correlates with distance between genes
Recombination events are more likely to occur between genes far apart on a
chromosome
A B C D
c d
(S. Triezenberg and J. Wang,
V. Germ cell division with crossing-over (recombination) in meiosis:
Parental genetic information needs to get resorted, and redistributed from generation
to generation. Offspring need to inherit genetic information derived from both parents,
but each parent has 2 copies of each gene from her/his parents to contribute. Thus, a
random reshuffling of genes occurs, with each parent only providing 1 of the 2
copies of each gene, in their gametes. This genetic reshuffling is accomplished
during prophase I, when each pair of chromosome homologs forms a bivalent, and the
homologs then exchange segments (genes), thereby reshuffling the genetic
information derived from both parents.
This exchange is referred to as crossing-over, and homologs can thus switch their
alleles. The closer the genes are on a chromosome the more likely they will be co
segregating (linked). In contrast, those genes that are far from each other on the
same chromosome, or on different chromosomes would be unlinked.
A bivalent of two homologs may also be referred to as a tetrad of four chromatids.

Only two non-sister chromatids exchange segments.
(J. Simmons, MSU)
Consequences of crossing-over in the tetrad following separation of homologs:
Crossing Over Meiosis I Meiosis II

(J. Simmons, MSU)
257
Recombination (crossover)
Takes place during pachytene of meiotic I prophase.
Results in an exchange of genetic material between paired homologs (bivalent).
The physical event, crossing-over, involves 2 of the 4 chromatids at any given
point along the length of the bivalent.
The farther apart any two genes are on a chromosome, the greater the likelihood
the crossing over will take place between them.
Crossing over is a normal and probably necessary step in meiosis: each
homolog pair must undergo at least one crossover event during each meiosis.
Absent (achiasmata) or reduced recombination has been found in 45% of
females who had Trisomy 21 offspring, but it is very rare in females meiosis
with normal offspring.
At the end of prophase of meiosis I, a chromosome pair which has undergone
one crossover event will consist of:
o 2 parental chromatids, one each of maternal and paternal origin,
o 2 recombinant chromatids, each containing both maternal and paternal
alleles in a complementary pattern.
(J. Simmons, MSU)
VI. Female vs. Male meiosis

A. Female meiosis begins prenatally (3rd to 4th month) and is arrested in the first
meiotic prophase in a special stage called dictyotene of diplotene, which is a stage that
only exists in female, in 9th month of fetal life. Meiosis I does not proceed until the
egg is ovulated 10-50 years later.
Female meiosis vs. male meiosis
Male Female
Commences Puberty Early embryonic life (3rd-
4th month)
Duration of meiosis 60-65 days 10-50 years (only complete
after fertilization)
Gametes/meiosis 4 spermatids 1 ovum and 2-3 polar
bodies
Gamete production 100-200 million per 1 ovum per menstrual
ejaculate cycle
B. Gametogenesis and fertilization
Stages o f oogenesis and spermatogenesis, n, haploid number.
Turnpenny: Emery's Elements of Medical Genetics, 14e

Copyright 2011 by Churchill Livingstone, an imprint of Elsevier Ltd, Al
1. Spermatogenesis
The spermatogonia develop from the primodial germ cells by a long series of about 200
mitoses.
Spermatogonium Primary Secondary Spermatid Spermatozoan
(46,XY) Spermatocyte Spermatocyte
(46,XY) (23,X, or 23,Y) (23,X or 23,Y)
Repeated mitoses
sustain this production M eiosis I M eiosis II
(P. Storto and R. Ritchie, MSU)
Human males produce 1000 sperm/second (30 billion/year).
Due to repeated replication of the chromosomes, there is an increased potential for
accumulation of minor chromosomal deletions/duplications over time.
Certain types of chromosomal disorders, such as achondroplasia, have been
documented.
2. O o g e n e sis: largely confined to prenatal development.

Oogonia have descended from the primodial germ cells by abut 30 mitoses.
(46,XX) (46,XX) (23,X) (23,X)
Oogonium Prim ary Secondary Oocyte Ovum &
(prenatal M eiosis I (Ovulation M eiosis II 2 pronuclei

developm ent) (at birth) triggers) (metaphase) (fusion into
[dictyotene stage] diploid zygote)
(P. Storto andR. Ritchie, MSU)
Women are bom with all of the primary oocytes they will ever have (~2
million), most degenerate. The primary oocyte have all reached prophase I by
the time of birth, and those that do not degenerate remain in that stage for
decades.
At puberty, 400,000 left. Only about 400 eventually mature.
Each month -1000 primary oocytes will attempt to mature, most will die.
Ovulation occurs 1 every -28 days. Females ovulate -400 times during their
lifetime.
^Chromosome abnormalities associated with advanced maternal age (i.e. aneuploidies

etc.) may be due to the long time they remain paired during Meiosis I (Prophase I).
3. C linical Relevance:
Non-disjunction: failure of the homologous chromosomes to separate during
meiosis I or sister chromatids to separate during meiosis II (or mitosis).
o Incidence:
More common in maternal meiotic divisions
More common in meiosis I
o Causes:
Aging effect on primary oocyte
- Absence of recombination (chiasmata) in prophase of meiosis I
- Abnormality in spindle formation
o Consequences: different chromosomes found in the gamete
Meiosis I: gamete contains both homologs of a chromosome pair
Meiosis II: two copies of one of the homologs of the chromosome pair
A B C
Disomic
gamete (Turnpenny 14F3.17_p43)
Turnpenny: Emery's Elements of Medical Genetics, 14e
Copyright 2011 by Churchill Livingstone, an imprint of Elsevier Ltd. All rights reserved.
Mitosis (zygote): two or more different cell lines

1 cell zygote
For sessions of 09/12/17, you should review Tutorial #2 (on nucleotides). The
nomenclature and structures of purines, pyrimidines, ribo- and deoxyribo-
nucleosides, as well as the corresponding nucleotides, will be critical in
understanding our discussion of their biosynthesis and catabolism.
ONE-CARBON METABOLISM and NUCLEOTIDE METABOLISM
In Session 18, we will discuss the biosynthesis o f purine and pyrim idine nucleotides. Because
nucleotide biosynthesis involves one-carbon transfer reactions, however, w e first need to do a
slight detour and discuss one-carbon m etabolism in Session 17. There are four im portant carriers
o f one-carbon units in am ino acid, carbohydrate and fatty acid metabolism : biotin (vitamin B7),
cobalam in (vitamin B12), folic acid (vitam in B9), and S-adenosylmethionine. The first o f these,
biotin, was discussed in the w ater-soluble vitam in lecture (BM B 515 session 6). The one-carbon
unit in tetrahydrofolate (THF) is directly related to the corresponding one-carbon unit in CH3-B12
and in S-adenosylm ethionine (SAM). THF plays a key role in nucleotide m etabolism and we
will describe the pathways o f synthesis o f purine and pyrim idine nucleotides, em phasizing the
steps at which regulation occurs. W e will also consider nucleotide catabolism and aberrations
thereof that cause disease.
Required reading:
Ferrier. 7ed: Chapter 20: A m ino Acid D egradation and Synthesis - from "Amino acids that fo rm
succinyl coenzyme A : methionine " up to "Other am ino acids that fo rm succinyl coenzyme A "
Suggested reading:
Ferrier. 7ed: Chapter 22: N ucleotide M etabolism - from "Synthesis o f purine nucleotides" up to
Fig. 22.24 "Key concept map f o r nucleotide metabolism"
Ferrier. 7ed: Chapter 28: V itam ins - from "F o lic acid" up to "Ascorbic a cid (vitam in )"
Objectives:
1. D escribe the im portance and the source o f the one-carbon carriers (SAM , CH 3-B 12, and THF
derivatives) and establish their relationship in term s o f the flow o f one-carbon units.
2. D escribe the biosynthesis o f purine and pyrim idine nucleotides from m etabolic interm ediates
and identify the key points o f regulation.
3. Trace the catabolism o f purine and pyrim idine nucleotides and identify the im portance o f
salvage pathw ays o f nucleotide regeneration
Measureable Outcomes:What You Should Be Able to Do....

I. Tetrahydrofolate(THF), cobalamin (vitam in B 12) and S-adenosylmethionine (SAM)
A. Identify the substrates, products, and tw o inhibitors o f the enzyme dihydrofolatereductase
(DHFR). (Obj. 1, 2)
B. D escribe the sources o f one-carbon units acquired by THF. (Obj. 1)
C. D istinguish the structures o f one-carbon units in various oxidation states as carried by
THF and describe how one can be converted to another. (Obj. 1)
D. Identify the dietary sources o f folate as a vitam in and describe the biochem ical and
physiological effects o f folate deficiency (m egaloblastic anemia). (Obj. 1)
E. D escribe the synthesis o f SAM, including substrates and position o f activated
methyl group. (Obj. 1)
F. Describe typical substrates and products for transfer o f methyl group from SAM and apply
this principle to a newly given substrate or product. (Obj. 1)
G. N am e substrates and products involved in regeneration o f methionine, including the
role o f cobalamin (vitamin B 12). (Obj. 1)
H. Compare the effects o f folate deficiency and vitam in B 12 deficiency. (Obj. 1)
I. Explain the methyl trap hypothesis for pernicious anemia, including the roles o f vitamin
B 12, intrinsic factor, m ethyl-THF, and methionine. (Obj. 1)
II. Nucleotide Synthesis

A. Identify com m itted and regulated steps in purine nucleotide biosynthesis, including the
crucial role played by 5-phosphoribosyl-1-phosphate (PRPP). (Obj. 2)
B. D escribe the roles o f PR PP synthetase and PRPP amido transferase in the purine nucleotide
biosynthetic pathway and their m ajor regulatory molecules. (Obj. 2)
C. Identify the carbamoyl phosphate synthetase II (CPS-II) reaction as the key regulated step in
pyrim idine nucleotide biosynthesis and name the regulatory molecules. (Obj. 2)
D. Distinguish tw o m echanism s that can lead to orotic aciduria in term s o f cause, consequence,
and m anagem ent options. (Obj. 2)
E. Describe the synthesis o f cytidine and thym idine nucleotides from uridine nucleotide
precursors. (Obj. 1, 2)
F. Identify the role o f tetrahydrofolate in the reaction catalyzed by thym idylate synthase and
the indirect involvem ent o f DHFR. (Obj. 1, 2)
G. Rationalize the im pact o f 5-fluorouracil and m ethotrexate on nucleotide pools and
(indirectly) on D N A synthesis. (Obj. 1, 2)
H. List the substrates and products o f ribonucleotide reductase and describe the roles o f ATP
anddATP in regulating the overall activity o f the enzyme. (Obj. 2)
III. Nucleotide Catabolism and Salvage Pathways

A. Trace the catabolic steps from AM P and GM P to uric acid, with particular attention to
the role o f xanthine oxidase. (Obj. 3)
B. Identify (in general term s) the substrates and products o f 5-nucleotidase and nucleoside
phosphorylase in the catabolism o f purine nucleotides. (Obj. 3)
C. N am e the substrates and products o f the m ajor purine salvage pathway, catalyzed by the
enzymes: hypoxanthine-guanine phosphoribosyl transferase (H G PRT) and adenine
phoshoribosyl transferase (APRT). (Obj. 3)
D. Com pare the role o f PR PP in the salvage versus d e novo pathw ays o f purine nucleotide
synthesis. (Obj. 2, 3)
E. Describe three types o f defects in the regulation o f purine nucleotide m etabolism that may
contribute to hyperuricem ia and gout, including the hereditary condition o f Lesch-Nyhan
syndrome. (Obj. 2, 3)
F. Com pare the biochem ical and physiological basis o f hyperuricem ia in term s of: (i) over
production versus im paired excretion; (ii) telltale signs o f uric acid levels in serum versus
urine; and (iii) treatm ent options (e g. allopurinol versus probenecid). (Obj. 3)
G. Identify catabolic products o f pyrim idine nucleotides and their fate in metabolism. (Obj.3)
C lin ic a l V ig n e t t e (on which the clicker questions at the end of Session 1 8 will be based)
ID/CC (identification and chief complaint): A 55-year-old Caucasian woman named LG; loss of
energy, easily fatigued; dyspnea (difficult breathing) on climbing two flights of stairs; weight
loss of eight pounds over the last six months; upon questioning, she remembers a few recent
episodes of paresthesia (tingling) in her toes.
HPI/ROS (history of present illness and review of systems): About nine years ago, her family
physician diagnosed her with anemia, which was treated with monthly "shots" (but she does not
know the content of those "shots"). She has had no "shots" in the last year because her family
physician died at that time and she has not yet connected with another family practice.
PE (physical exam): Vital signs, normal. Skin, pallor (pale coloration of skin). Otherwise
unremarkable.
Labs: CBC (complete blood count) results

(a) HCT (hematocrit) LOW (relative to normal range for females, 37-47%);
(b) RBC (red blood cell count) LOW (normal range for females, 4.2-5.4 x 106/pl);
(c) HGB (plasma hemoglobin concentration) LOW (normal range for females, 12-16 g/dl);
(d) MCV (mean corpuscular volume) HIGH (normal range, 80-96 fl)
(e) blood smear revealed macrocytic RBCs and hypersegmented PMNs (polymorphonuclear
leukocytes = neutrophils)
Big Picture for today's discussion on One-Carbon Metabolism
I. One carbon (1C) units:

Methyl: -CH3
Methylene: -CH2
Methenyl: -CH
Formyl: -CHO
Formimino: -CHNH
II. Flow of 1C unit transfer

Carrier Acceptor plus 1C
lC-donors: (serine, <
(Products: serine, glycine,
glycine, tryptophan,
histidine, formate) m ethionine, thymidylate,
purines, methylated
compounds)
donors Carrier
minus 1C plus 1C Acceptor
III. THF, V ita m in B 1 2 , SAM fu n c tio n as c a rrie rs f o r 1C u n it tra n s fe r
THF = te tra h y d ro fo la te
M et = m e th io n in e
SAM = S -a d e n o s y lm e th io n in e
S A H C = S -a d e n o s y lh o m o c y s te in e
I. Tetrahydrofolate (THF)
A. Structure and synthesis of THF and derivatives Harvey5 _F2 8 .3 _ P3 7 4
Humans and
Microorganisms microorganisms
HjN Amino acid

2 NADPH
+ 21-T ' 2NADP yj synthesis
CH, Dihydro- Olhydro-
OH
I HjN COOH pleroate
synthetase
folate
reduetasej
p-Aminobenzoic acid Folic acid Tetrahydro- Purine
Pteridlne precursor fo lk acid
(PABA) synthesis

A A
Sulfonamides Methotrexate Th ym idine

(used as anti- (used to treat synthesis
microbiat agents) acute lympho
cytic leukemia)
Folate Dihydrofolate (DHF) Tetrahydrofolate (THF)

(vitamin) (coenzyme)
1-C units at
different methionine
oxidation synthesis
states
AT-Matfiyt-
source of tati i tiydrofotm
1-C unit most

reduced
thymidine synthesis
nucleotides for
hc----M-
DNA and RNA
W1Q-Formyl-
Ulrahytk'OWat
most C-8 of purine
nYirli7P>rl C-2 of purine
B. Folic acid as a vitamin (vitamin Bg)
1. present in wide range of foods (green leafy vegetables; liver; lima beans)
also produced by bacteria in intestine.
2. yet, it is probably the most common vitamin deficiency in the US

pregnant women increased demand;
alcoholics absorption problems;
patients undergoing chemotherapy folate to THF conversion inhibited
3. effects of deficiency
THF is used in 1-C transfer reactions, particularly important in
nucleotide synthesis (for DNA and RNA).
a)
- rapidly dividing cells in hemopoiesis

require DNA and RNA synthesis;
- precursors to erythrocytes enlarge
but do not divide; the larger than
normal precursor cells lyse.
Complete Blood Count (CBC)

[details in PSL 539 Session 18]
hematocrit (HCT): LOW (f: 37 - 47 %)
red blood cell count (RBC):
LOW (f: 4.2-5.4 x 106 /pl)
plasma hemoglobin concentration
(HGB): LOW (f: 12-16 g/dl)
mean corpuscular volume (MCV):
HIGH (80 - 96 fl)
- also seen in pernicious anemia due
Ferrier7 F28.4 to vitamin B12 deficiency
b) Neural tube defects: "open spine neural tube birth defect due to
defective spinal column closure; folate critical for development during first
weeks of fetal life; dietary supplementation to cover woman of childbearing
age (before pregnancy is even suspected)
P erico n cep tio n al folate in ta k e an d n e u ra l tu b e defects.
R ay b u rn ,-W -F ; S tanley,-J-R ; G a rre tt,-M -E
J-A m -C o ll-N u tr. 1996 A p r; 1512): 121-5
A b stra ct: Approximately 50% of neural tube defects may be folate-preventable and perhaps
even more in other countries where prevalence is high. The Public Health Service has issued the
recommendation that all women of childbearing age in the United States who are capable of
becoming pregnant should consume 400 micrograms of folic acid/day for the purpose of
reducing this risk._________________________________________________________________
P lasm a F olate Levels an d R isk of S pontaneous A b o rtio n

Lena George, MD; James L. Mills, MD,MS; Anna L. V. Johansson, MSc; Anna Nordmark,
MSc; Bodil Olander, MD; Fredrik Granath, PhD; Sven Cnattingius, MD,PhD
JAM A. 2002;288:1867-1873.
C onclusion Low p lasm a folate levels w ere associated w ith an increased risk of early
spontaneous abortion.
C. Drugs that affect THF metabolism

Humana and
Microorganisms microorganism*
Amino Add
2 WAOPH synthesis
2 KAOP
P^yCtO-
cooh plewala
* o Synthefjso
Purina
p-AminoPemoreat'1
d F o Jit JCKJ T n tra h y d ro -
M i c a c id s y n th e s is
[PABA)
Methotrexate Thymidina
SvJffcjnamides s y n th e s is
** anti- (used to trust
(mcroWal agents) a cu te ly m p h o
c y tic le u ke m ia l
Harvey5_F28.3_p374
1 .methotrexate (and aminopterin)

- structural analogs of folic acid H
- inhibitors of DHFR
- used as an anti-cancer drug to
inhibit DNA synthesis of rapidly
dividing tumor cells
2. trimethoprim(TMP) and pyrimethamine

- also inhibitors of DHFR, but more specific for bacterial DHFR;
- can be used as an antibiotic;
- used for treatment of malaria and Toxoplasma gondii infections.
3. sulfonamides (sulfa drugs) structural analogs of PABA

- inhibit bacterial dihydropteroate synthetase
- used to inhibit bacterial synthesis of folate (and THF)
- no corresponding enzyme in humans (therefore, good antibiotic)
- however, sulfa drugs can produce oxidant stress and aggravate
glucose 6 -phosphate dehydrogenase deficiency (like anti-malarials)
4. Sulfamethoxazole/ Trimethoprim (SMZ/TMP) -combination of sulfa

drug and trimethoprim
- antibiotic action specific against bacterial THF synthesis.
[If you prescribed sulfa drugs to treat an urinary tract infection in a female patient, would
your thinking change if she were pregnant?.... if she were of child-bearing age?]
II. S-adenosyl Methionine (SAM, AdoMet)
A. Activation of methyl group of methionine

NHj
COO
COCr I
I H ---- C N H 3*
H C N H 3 I
I CH,
CH, I
I CH,
CH, I
I h3c s ch2 adenine
h3c s
M ethionine
ATP { fsi
OH OH
<6> S-A dorvosytm othionina
B. Transfer of activated methyl group

Principle:
R------OH R------ O ----- CH)
<f R------ NHj r ---- N H ---- CH) COO COO
I HzO Adenosine
I
h c NHi. y y H NHu
I ->~ H C NHv
CH CHj CH2
I I
CH O-tj CK>
K jC S Adenosyl S Adenosyt
I
SH
*
S-Adenoeytmethionine S-A denosyl-
H o m ocysteine
hom ocystolrx)
Application:
o
O 1CHj o o R,
II 2
R,0 -0 CH O
X H j-0 P- -CHj-CHzNHz
I
C Ethanolamhtf
CboGre
3-Phosphatidylethanolamine
3-PhosphatkfytehoTtfve
norepinephrine-------------epinephrine (CH3 group added to NH2)
epigenetic modification of DNA

(methylation of the base C in 5'CpG3' islands) (supplemental reading)
III. Vitamin B 12 links the 1-C unit in THF and in SAM
N5-methyl THF
methylene
THF
This is the only rxn that N5-methyl-THF can undergo
methyl trap hypothesis: (i) if no B1 2 , N5 -methyl-THF stuck;

(ii) since the reaction from N5, N1 0 -methylene-TFIF to N5 -methyl-THF is
irreversible, the THF "skeletons" are trapped in the form of N5 -methyl-
THF, whose levels rise high above normal (while the levels of CH3-B12
and Met are lower than normal);
(iii) not enough THF in other oxidation states for nucleotide biosynthesis
deficiency=> deficiency (and megaloblastic anemia)

AND deficiency as well.
Therefore, there are consequences both upstream and downstream.
D. Megaloblastic anemia due to vitamin B1 2 deficiency (problems in
addition to folate deficiency)
animal origin (liver, milk, eggs, etc.)
2. Absorption of vitamin present in diet

requires intrinsic factor (IF)
which binds B12 in the stomach and
mediates its absorption into the intestinal
mucosal cells via a receptor specific for IF.
If B12 deficiency is suspected, need to test

for both amount of B12 in the body and for
absorption.
Schilling Test: radioactive B12 IF.
(a) treat with B12 only (no exogenous IF)
if B12 absorbed, true B12 deficiency
(b) B 12 with exogenous IF if B 12 is now
absorbed, most likely IF deficiency
(c) if neither of the above works, then
most likely a problem of the IF receptor
[Most known B12 deficiency in humans can be
accounted for by absorption problems. Given this,
what kinds of information regarding your patient
would be critical to consider if you suspect an
apparent B12 deficiency?]
Ferrier7 F28.7
3. B12 deficiency leads to folate deficiency (methyl trap hypothesis).
Apparent megaloblastic anemia should be treated with supplementation in:

vitamin B12 folate SAM
4. In addition to anemia due to folate deficiency, however, other problems:

a)methionine deficiency - but Met can be derived from less prominent
alternate routes
choline (diet) glycine ------donate C H 3 ------Met
betaine ------ to homocysteine
H o m o c y s t e in e N 5- M e t h y l-
te tra h y d r o fo la t e
Methionine Vitamin B 12 a) Already discussed: vitamin B12

synthase (Methyl-
cobalamin)
links the 1-C unit in THF with Met
V and SAM
T e t r a h y d r o f o la t e
M e t h io n i n e
F a tty a c id s
(od d n u m b e r o f c a rb o n s )
b) CH3-B12 involved in other
metabolic rxns methylmalonyl
CoA succinyl CoA (TCA cycle)
COO'
H3C - C - H
(i) abnormal fatty acids in
t '
C C -C oA membrane structure of nervous
6 system skin sensation tingling,
M e t h y lm a lo n y l C o A prickling, itching such neurologic
Methylmalonyl CoA Vitamin B 12 problems are irreversible
mutase (Deoxyadenosyl-
y cobalamin)
COO'
(ii) methyl malonate accumulation------
h 2c -ch2
methylmalonyl aciduri (acidosis)
C -C oA
11
o So, pernicious anemia due to B12
S u c c in y l C o A
deficiency exhibits symptoms (e.g.
C o p y rig h t 2008 W o lte rs K lu w er H ealth |Lip p in cott W illiam s & W ilkins
neurological problems) in addition
Ferrier7 F28.5 to megaloblastic anemia seen with
folate deficiency.
I. Big Picture Overview of Nucleotide Metabolism Ferrier7_F3o.2
Dr. Thomas Traut (Mol. Cell Biochem. 140: 1-22 (1994) com piled inform ation from some 600
publications on the physiological concentrations o f purines and pyrim idines. The inform ation
sum m arized below puts our forthcom ing discussion in some perspective.
Ribonucleotides (NTPs) Deoxvribonucleotides (dNTPs)
ATP -3 ,0 0 0 pM dATP -2 4 pM
GTP 470 dGTP 5 [In general, dNTPs are 10 to
CTP 280 dCTP 29 1000-fold LO W ER than NTPs;
UTP 570 dUTP 0.2 don't w ant to m ess around with
dTTP 37 genetic material ]
1) The average concentration o f bases and nucleosides in plasm a and other extracellular
fluids is in the range o f 0.4 - 6 pM (low er than intracellularconcentrations)
2) O ther cellular concentrations o f interest: (a) Pi, -4 ,4 0 0 pM ; (b) ribose 1-P,~55 pM;
(c) ribose 5-P, ~70 pM; and (d) 5-phosphoribosyl-l-pyrophosphate(PR PP), ~9 pM.
3) Tum or cells have 6-11 tim es more dNTPs than normal cells; tum or cells have
1.2-5 tim es m ore N TPs than normal cells
II. Purine nucleotide biosynthesis
fl
*op - och2 Strategy: -assemble purine ring de novo or through
'O salvage pathway
- attach to ribose-phosphate -> IMP
HO OH - modify to form other purine nucleotides
ribose 5-phosphate
ATP v ACTIVATION STEP
PRPP synthetase
AM ?*
regulated: ATP (key regulator)
OCHj GMP (end products
AMP of pathway)
OH OH
5-phosphoribosyl-1-pyrophosphate Intracellular [PRPP] (availability)

G in ------ critical in regulating rate of this step;
Glu,
PPi
[PRPP] ~10"5 to 10~6 M;
COMMITTED STEP
PRPP amido transferase; Km ~ 10~4 M
regulated: IMP, GMP, AMP
HO OH
(all end products of pathway)
5-phosphoribosyl-1 -amine
assemble purine ring de novo 9 steps, involving C 0 2, Asp, Gin, Gly,

CO; Glycine
and N10-formyl THF (p. )
Aspartate ^
lS donating C and N atoms of purine rings
n a c ox.
ac -
u c ^ a ,c ^ o 7
N N
uses 4 more ATPs
t_ N
1 Glutamine
N10-Form yl- --------- FSbose
tetrahydrofolate
inosine monophosphate
H
OOO C l fcC ~ COO"
Ferrier7 F22.5 I
NH
>
1
H Rfcose <P> Ribose
xanthine monophosphate adenylosuccinate
guanosine monophosphate adenosine monophosphate

III. Pyrimidine nucleotide biosynthesis
A. Strategy: - assemble pyrimidine ring
- attach to ribose-phosphate -> UMP
- modify to form other pyrimidine nucleotides
B. De novo synthesis:
1 ) reaction pathway
H zN ^5 UTP
i
9*2 (end product)
9Hb
+h3n -< |-h carbamoyl phosphate
co* synthetase II (cytosol)
if /
GMsunlra UDP
<^>-0-042 <f<X>r'CH
ATP HCO3
PRPP Glutamata I O I
UTP wo
HjjNOOPOg2* OH
Urtdla. S-monopho.pt,a u <UMP)
qqq- Carbamoyl phosphate

I HCOg^
+H3N
H jO -
coo* O
II
Aspartate Pf- ntf'SDM
O- o
II
HzN l > ---- r Orotate monophosphate (OMP)
Carbamoyl aspartate
HO OH
/ S r ' ' cc-
PPi
orotate phosphoribosyl
jPRPP|' transferase (OPRT)
H?o
o
II
H
D lhydroorotate 7 ^ \ i
NAD* NADH + H*
H
OroUt.
2) Orotic Aciduria excessive orotate in the urine

a) OPRT deficiency:
i) decreased availability of pyrimidine nucleotides for DNA
and RNA synthesis megaloblastic anemia
HOT: low; HGB: low; RBC: low; MOV: high
ii) Feeding uridine (by-passing the deficient enzyme step)
uridine (nucleoside) UMPÛDPÛTP (and CTP)
alleviates the anemia and provides inhibitor of CPS-II.
b) Urea cycle defects:
i) carbamoyl phosphate is also produced by the mitochondrial
isozyme carbamoyl phosphate synthetase I (CPS-I) for the
urea cycle (which functions in the liver to handle nitrogen from
amino acid and protein metabolism Semester 3b)
ii) if there is a problem with urea cycle, accumulated carbamoyl
phosphate will spill into the pyrimidine biosynthetic pathway,
leading to orotic aciduria (but not anemia because pyrimidine
nucleotides are still synthesized OK).
C. Roadmap from UMP to other pyrimidine nucleotides

Parenthetical remark: kinases use ATP to add phosphate groups;
illustrated here for U (uracil) but also true for A, G, C, and T as well.
Nucleoside kinase : e.g. uridine kinase; adenosine kinase; thymidine kinase

uridine (nucleoside) UMP (nucleoside monophosphate = nucleotide)
Nucleoside monophosphate kinase and Nucleoside diphosphate kinase

UMP udp ------- utp ------- C T P
i
dU D P ---- - dUM P ---- dT M P
D. Synthesis of cytidine triphosphate (CTP)

O
U TP CTP
IV. Deoxynucleotide Biosynthesis

NADPH NADP*
A. reactions
Ribonucleotide
reductase
B. dUMP dTMP
1. Pathway
Methylene THF
THF --------
H s. N 10- IMftylefM- 7,MMhydrofotat
,---NAOPH.H.
CHFFI
$tnt - ^-
NAOP.
O V.
5 - F lu o 1 0 u i*a c i l
Tetrehvdreietete
2. Inhibition by 5-fluorouracil (5-FU) as an anti-tumor agent
a. F, instead of H, at C-5 (cannot substitute CH3 group at C-5);

FdUMP (instead of dUMP) binds to substrate site of enzyme TS;
permanently stuck on enzyme and depletes active enzyme TS;
low production of dTMP (and thus, low dTTP for DNA synthesis)
b. Rapidly proliferating cells (tumor cells) need supply of dTMP for DNA
synthesis; proliferation of 5-FU-treated cells inhibited.
c. 5-FU can be used in conjunction with methotrexate

[What is methotrexate?
What enzyme does it inhibit?
Due to this inhibition, the availability of is lowered,
thus depriving the enzyme of its substrate.
d. Both 5-FU and methotrexate have the same overall effect: little dTMP
produced and thus the combination can be used to inhibit the proliferation of tumor
cells in chemotherapy.
BMB515
V. Nucleotide Catabolism
A. Catabolism of purines - uric acid
HjO NHj
AMP* J O . IMP GUP
nucleotide
Hjp
[ S'-Nodaolfala
Pt- K<0 NH,
P|..
A 4 * n * ln f 1 _ - I n o a tn * nucleoside GMitoaln*
X a n th ln *
The enol form is weakly

acidic (can dissociate a +HjO
H+to yield urate).
pH of serum: ~7
predominant form
is urate
pH of urine: ~5
predomiant form
is uric acid
(very low solubility)
crystallizes out
B. Pyrimidine catabolism similar idea: nucleotide^nucleoside^base

The pyrimidine rings are converted to intermediates of carbohydrate and
lipid metabolism (malonyl-CoA; succinyl-CoA).
VI. Salvage pathways for purine bases
A. the notion of "salvage" to capture the free base and prevent
further breakdown by returning it to the ribose 5-P foundation
B. Salvage reactions ---
note PRPP is substrate and thus, product is always a ribonucleotide
1) hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
PRPP + hypoxanthine (or guanine)------ IMP (orGMP)
2) adenine phosphoribosyl transferase (APRT)
PRPP + adenine------ AMP
VII. Hyperuricemia and gout
A. Uric acid (even as urate) is poorly soluble in aqueous medium

Normal serum concentrations: 4.5-8 mg/dL (m); 2.5-6.5 mg/dl_ (f)
B. Hyperuricemia: high serum levels ( 7 mg/dL) crystallizes out in
capillaries and joints >pain
birefrigent urate crystals in PMN leukocytes tophi (crystal deposition)
Ferrier7 F22.18 Ferrier7_F22.16

C. Causes of gout:
1) physiology: diminished renal excretion of uric acid
Excretion depends on an anion transporter in the kidney; uric acid

competes with anions of other organic acids (e.g. acetoacetate, etc.)
Uricosuric drugs (such as probenecid, benzbromarone, and sulfinpyrzone)

are to keep serum uric acid levels low. They promote increased excretion
of uric acid by inhibiting its re-absorption in the kidney (urine retains more
uric acid, thus lowering its concentration in the plasma).
2) Biochemistry: overproduction of purines
hyperactive PRPP synthetase

([PRPP] availability determines the rate de novo purine synthesis)
decreased feedback inhibition of committed step
reduced HGPRT activity
Adenine
PRPP
APRT
PPi
Ribose .AMP -ATP
5 -p h o s p h a te
+ ATP ------- -PRPP-----------Phospho- _
PRPP ribosylamine
-IM
PRPP
synthetase am ido-
transferase 'GMP -GTP
0
Loss of PP,
Elevated
feedback
levels
inhibition hgprt *N
Decreased levels
0
PRPP
Hypoxanthine Guanine
Uric acid
If HGPRT i, salvage j; hypoxanthine and guanine t, uric acid t-
More importantly, PRPP not used in salvage; stimulate de novo purine
nucleotide synthesis > more for degradation to uric acid.
D. Biochemical approach to treatment of gout
1) Inhibitor of xanthine oxidase (e.g. allopurinol; febuxostat)
Hypoxanthioo
Guanine
purine: N7, C8 iiloputlno)

allopurinol: C7, N8
permanently stuck on xanthine oxidase (suicide inhibitor)
Unate
less uric acid, more xanthine and hypoxanthine

increased salvage of H and G, using up PRPP
salvage of allopurinol (structurally just like a purine base)
uses up PRPP (less for de novo purine synthesis)
E. Lesch-Nvhan syndrome: hereditary lack of HGPRT

1)metabolic consequences
(i) salvage (ii) de novo synthesis (iii) uric acid
less IMP & GMP to feedback inhibit more PRPP available hyperuricemia
2)phenotypic consequences (symptoms)

(i) gout
(ii) neurological problems

mental deficiency
spasticity (involuntary movements)
self mutilation (biting of lips;
chewing fingers down to the bones)
3)allopurinol therapy alleviates gouty

symptoms but not neurological problems
Ferrier7 F 2 2 .ll
F. Serum versus urinary uric acid
Remember: it is the crystallization of uric acid in serum that causes the

problems in the body. A comparison of uric acid in serum and in urine can
be informative:
1) HIGH, both in serum and in urine ---

increased risk for gout (because high serum concentration)
most likely due to overproduction (biochemical problem)
kidneys appear to be dumping as much as possible (high in urine)
2) HIGH in serum but not in urine ---

increased risk for gout (because high serum concentration)
most likely due to an underlying renal problem
kidneys should be dumping the large amounts of uric acid in blood but is
apparently not doing so (not high in urine)
3) HIGH in urine but not in serum

not at risk for gout (because serum concentration is OK)
this is most likely someone taking a uricosuric drug to increase
excretion (high in urine)
Supplementary Reading: epigenetic modification of DNA
(methylation of the base C in 5'CpG3' islands)
Un methylated
NH2
Methylated
cytosine
cytosine
O
5 0
CH2
O H
3
5-CpG-3'
There appears to be sweeping epigenetic modifications taking place through the germline during
development. The oocyte and the somatic cells of the ovarian follicle and pre-implantation
embryo are particularly vulnerable to alterations in one-carbon metabolism. Maternal dietary
status of folate, vitamin B 12 , and vitamin Be affect gene expression (through DNA methylation)
in the peri-conceptual period. The relationship between folate (B9 ) and B 12 was discussed in this
lecture. Vitamin B6 is also in this picture because the transfer from of 1-C unit from serine to
THF to form N 5, N 10-methylene THF is a vitamin B(,-dependent reaction.
K.D. Sinclair et al. (2007) Proc. Natl. Acad. Sci. USA 104: 19351-19356
W.Y. Kwong et al. (2010) Reproduction 139: 705-715
DNA methylation may affect over-expression of Insulin-like Growth Factor-2 (1GF-2). This
effect appears to be particularly noticeable in the male offspring...an example of genetic
imprinting such as seen with Beckwith-Weidemann syndrome (visceromegalylarge liver and
spleen; hemi-hyperplasia one part of body bigger; body proportion not right). Insulin
resistance and blood pressure in the offspring are also affected.
Offspring from in vitro fertilization (Assisted Reproduction) have a higher frequency of

disorders due to abnormal imprinting than the general population. Examples include increased
incidences of Beckwith-Weidemann syndrome, Prader-Willi syndrome, and Angelman syndrome
(all coming up in BMB 527). The inference is that embryos cultured for in vitro fertilization are
exposed to concentrations of methionine, folate, B 12 and B6 in the culture medium that are quite
different from endogenous in vivo concentrations of such compounds. This may affect DNA
methylation and imprinting.
RNA Transcription
In this lecture, we will look at the structure and the various functional
classes of RNA. First, we will examine the essential features of the RNA
molecules in general, then the biosynthesis (or transcription) process in E.
coli. Next, we will explore transcription and the subsequent processing
events in eukaryotes.
Suggested Reading:
Ferrier: Chapter 31: RNA Structure, Synthesis, and Processing - from Overview"
through Chapter Summary"
Turnpenny: Chapter 2: The Cellular and Molecular Basis of Inheritance - Transcription

(p. 18-19)
Objectives:
1. Understand how the diversity of observed RNA structures reflects the diverse and
complex functions of different classes of RNAs (ribonucleic acid, G:U basepairs mRNA,
rRNA, tRNA, hnRNA, snRNA).
2. Understand the process of RNA biosynthesis in bacteria and eukaryotes

(transcription, promoter, sigma factor, RNA polymerase I, RNA polymerase II, RNA
polymerase III, 5' cap, 3 polyadenylate tail, splicing, intron, exon, snRNP, spliceosome,
splice site).
3. Understand the role and biological significance of RNAs in gene regulation and
catalysis (gene silencing, short interfering RNAs, microRNAs, RNA editing, catalytic
RNA).
1. Recognize and describe the structures of the four common bases and the sugar
found in RNA. (Obj. 1)
2. Describe and read the primary structure of RNA, and recognize the 5' and 3' ends of
an RNA shown in a structure diagram in order to determine directionality. (Obj. 1)
3. Describe the participation of RNA in double-stranded nucleic acids, including G:U

basepairs. (Obj. 1)
4. Describe general functions, relative abundance, and complexities of the following

classes of RNA: mRNA, rRNA, tRNA, hnRNA, snRNA. (Obj. 1)
5. List the essential components of a transcription reaction in bacteria and eukaryotes.
(Obj. 2)
6. Describe the subunit structure of E. coli core polymerase and holoenzyme. (Obj. 2)
7. Define promoter, and describe the function of sigma factor. (Obj. 2)
8. Describe the steps that occur within each of the three stages of transcription in
bacteria (initiation, elongation, termination). (Obj. 2)
9. Recognize transcription as a site of action for antibiotics. (Obj. 2)
10. Define the roles, relative activities, and locations of the three eukaryotic RNA
polymerases. (Obj. 2)
11. Describe the general features of a promoter for RNA polymerase II, in contrast to a
bacterial promoter. (Obj. 2)
12. Describe the role that basal and regulatory transcription factors play in eukaryotic
RNA synthesis. (Obj. 2)
13. Describe the events in processing of hnRNA into mRNA, including:

1. the structure and role of the 5' cap.
2. the structure and role of the 3' polyadenylate tail.
3. splicing, including
a. define exon and intron.
b. describe the general roles of snRNPs and the spliceosome.
c. define what is meant by 5' and 3' (or donor and acceptor) splice sites,
and describe the importance of the sequences at those sites. (Obj. 2)
14. Define the general concept and results of RNA-based gene silencing. (Obj. 3)
15. Distinguish between the origins of short interfering RNAs and microRNAs (Obj. 3)
16. List biological phenomena in which RNA silencing occurs. (Obj. 3)
17. Describe the technological and potentially therapeutic application of RNA silencing.
(Obj. 3)
18. Describe how RNA editing leads to increased variation in the human proteome.
(Obj. 3)
19. Define what is meant by the phrase "catalytic RNA," and give two examples. (Obj. 3)
I. RNA Structure
A. Components
Sugar: Ribose - 5 carbon sugar numbered
clockwise from the N-glycosidic bond, T through 5.
Contains a hydroxyl (OH) group at the 2 position,
which distinguishes Ribonucleic Acid from
Deoxyribonucleic Acid.
Bases: Purines - Adenine (A) and Guanine (G)
Pyrimidines - Cytosine (C) and Uracil (U) - has a hydrogen (H),
not a methyl group (CH3) at position 5 of the pyrimidine ring.
Primary Structure: Sequence of bases as read from
the 5 end of the molecule toward the 3 end.
NOTE: The terminal phosphate of the
backbone can be written at either end and it
DOES NOT change the polarity!
Secondary Structure: RNA is usually single
stranded, though it is flexible enough to fold
back on itself and form secondary structures,
often a critical component of the function.
The most basic secondary structure is a
Stem Loop (or a hair pin)
a' u- G
5' - G - C -A - U - G - A -U - C - G - C / \
/c
III ill II II III II II III II III
a ^ C -G -U -A -C ~ U -G -G -U -G x
u
NOTE: Within these structures, unusual base pairing of guanine with

uracil is allowed - this pairing is unique to RNA.
B. Major Functional Classes of RNA
1. Ribosomal RNA: (rRNA) is a major component of ribosomes,

which are part of the protein synthesis machinery.
Relative Abundance: rRNA accounts for 85% of the total RNA
within a cell, making it the most abundant class of RNAs
Size/Nomenclature: rRNA is named based on its size. This

size is a 3-D size in space (accounting for the shape) and is
noted in units of Svedberg (S), which is simply a sedimentation
rate through a defined medium by centrifugation. This is NOT
A LINEAR relationship, though in general, the larger the S
value, the higher the molecular weight.
For Example: (Do Not Memorize...)
Bacterial rRNAs 5S 16S 23S

(120 nt) (1540 nt) (3000 nt)
Eukaryotic rRNAs 5S 18S 28S 5.8S

(120 nt) (1900 nt) (3700 nt) (160 nt)
Base Composition: A, C, G, and U (with some modified bases)

Structure: Very complicated. Some even mimic protein
structure and exhibit enzymatic activity!
2. Transfer RNA: (tRNA) is a critical

component of protein synthesis and 3'-End
acts as a magic decoder ring Site of amino CCA
acid attachment JL
bridging the gap between nucleotide
sequence and protein sequence. 5'-End
Complementary
base pairs
Relative Abundance: tRNA (intrachain)
accounts for 10-12% of the total
RNA within a cell. Our cells have
56 tRNAs
(Ferrier7_31.3)
Anticodon
Size: tRNAs are of basically uniform size ranging from 75 - 90
nt in length.
Base Composition: A, C, G, and U with many modifications
Structure: 2D- is often called a clover leaf due to the way it is

commonly drawn (see above). Starting at the 5 end...
D-loop: named because it contains dihydrouracil
Anticodon loop: contains the anticodon which base pairs

with codons of mRNA.
The first position (5) of the anticodon (wobble
position) often contains a modified base
TwC loop: sometimes just called the T loop. Named

because it contains the base thymidine (the only RNA
species to do so). It also contains pseudouracil.
Acceptor Stem: (not coded for!) the 3 CCA stem is added

to every tRNA after transcription. ... more on this in the
processing section. This is where the amino acid will
attach to be carried to the growing peptide chain.
3D- common L-shaped structure for all tRNAs

3. Messenger RNA: (mRNA) is the middle man taking the genes
coded in DNA in a readable form to the ribosomes in order to
make protein.
Relative Abundance: 3-5% of total RNA in a cell, however these

molecules are specific for the proteins they encode, so the
abundance of any given mRNA is extremely low! There are 1 -
10 thousand (103-104) different mRNAs in a cell!
Size: highly variable, just like the genes that encode them.
(200 nt up to 104nt)
Base Composition: A, C, G, U with very little modification
Structure: The messages are structured in terms of an Open

Reading Frame (ORF) which is the region of a gene or an
mRNA that actually codes for the amino acid sequence of a
protein.
Polycistronic mRNAs are those that contain more than

one ORF and are found in bacterial systems and some
protists.
5I lr ..... " ?! ly*%iWMWy>|il I

ORF 1 ORF 2
Eukaryotic systems use a Monocistronic mRNA that
contains only one ORF which codes for one polypeptide.
5 Cap-[ Open Reading Frame . AAAAAAAA,n

Summary: Class Abundance Diversity

rRNA +++ 1
tRNA ++ 35
mRNA + 10,000s
II. Bacterial versus Eukaryotic Gene and Transcript - identify 2
things that are similar or different between bacteria and eukaryote
DNA Gene Prokaryote

5'- :3
+1
Transcription Transcription/
start Processing
Mature mRNA
5'pppG UUU 3'
DNA
>-1000 -40 to -150 -30^ 100 -3to +5 Gene Eukaryote
+1
Transcription
start Transcription/
Processing
Mature mRNA
5' m7Gppp -(AAA)n 3'
(M. Faner, MSU)
RNA Biosynthesis in E. coli (Transcription) based on a

template, usually DNA
A General Features:
Requires a DNA template, ribonucleoside triphosphates (ATP,
CTP, GTP, and UTP) and an RNA polymerase
Synthesis of RNA proceeds 5 3
NO Primer - this allows RNA to serve as the primer for DNA
synthesis!
Possible proofreading, though it is limited and not entirely
necessary because the product is not passed on to progeny.
Further, it generally has a short V2 life... it is used and
destroyed.
Template is conserved, it is neither used up nor changed
Process - overview:
o dsDNA opens to form a bubble
o As RNA is made, it stays paired with the DNA
o As the polymerase moves on the DNA duplex recloses,
the bubble moves to the right, and the RNA extends out
beyond the DNA-polymerase complex.
Product RNA strand is complementary to the template DNA
strand (also called the antisense strand, and it reads the same
as the non-template DNA strand (also called the coding strand
or the sense strand)
/ \ _____ i
X 5 _.3 / ------------ T.
3'
B. RNA Polymerase
In E. coli, there is only one RNA Polymerase (RNAP)
1. Structure:
Core polymerase (multisubunit) is catalytically active, but does
not know where to start transcribing... its a big genome out
there.
Holoenzyme is the core polymerase plus Sigma (a) factor.
This additional subunit directs the polymerase to the start site of
transcription.
- Rationale for needing to know where and how often to start

transcription: RNA polymerase only copies the bit of information
needed for that cell at that moment. Unlike DNA replication which
needs to copy the whole genome for passage onto progeny.
2. Reactions of RNA Polymerase

Initiation: START
Elongation: GO
Termination: STOP
a. In itia tio n : In order to start at the correct location, the RNA
polym erase holoenzym e recognizes a Promoter.
P rom oter: is a sequence elem ent (can have variation) that is

recognized by the a factor and directs binding of the RNAP. It is
usually upstream of the + 1 start site. The + 1 start site is represented
w ith a broken arrow. The variation in the prom oter elem ents can
change the strength of the promoter. [N ote : the gene sequence tells
the cell what to make, but the p ro m o te r tells the cell when to m ake it
and how much to make.]
S tra n d S eparation: RNAP RNA polymerase

holoenzyme
induces the DNA strands to -35 -10 +1
DNA
open, hence it has helicase
activity. Binding to promoter
Form ation o f 1st bond:

RNAP reads the sequence
and places the 1st nucleotide
(usually A TP ) and the 2 nd and
catalyzes form ation of that
first phosphodiester bond.
P ro m o te r E scape: The a
factor is released and left
behind and the core
polym erase alone carries out
elongation.
b. Elongation: Continuing synthesis of the new RNA strand by
the core RNAP.
Synthesis proceeds in a 5 3 direction at a rate of 35 - 50 nt per

second! This requires that the DNA be unwound ahead of the
growing RNA strand and that it be rewound once the polymerase
is past a particular point. Further, there is no significant
proofreading of the placed nucleotides.
RNA product is complementary, and base pairs with the template

strand of the DNA for about 10 - 12 nt.
c. Termination: Ending transcription at the right place is also

important
1
and is achieved in a /
^ >.
u (\ \
,PT. u
(S. Tnezenberg and
unique way. Lc ga J. Wang, MSU)
\ A A. /
A signal exists in the DNA

- G E. C
sequence, is transcribed into G+C-rich -C e G
hairpin | -C e G -
an RNA sequence, and is -C = G
consecutive Us
functional as an RNA -G = C
-C = G
i
STRUCTURE. RNA
TT IIIII
G-C rich sequence, which forms a stem-loop structure in the

RNA, followed by poly U, this affects the RNAP and causes it to
pause. The new RNA is released by terminator proteins, so it is
no longer base paired, and it falls away.
RNA:DNA hybrid
3. Inhibition by Antibiotics: This is one reason J No d rug prese n t
why we care! Understanding the biochemistry

of microbes allows us to mess with it!
Antibiotics can inhibit RNAP by several modes of

action which we will group into two classes: R N A p o ly m e r a s e
a. Inhibition by DNA-binding R ifam pin present
o. a
Actinomycin D = dactinomycin =
Cosmegen: This drug acts by blocking the
unwinding of DNA by RNAP. Used as an
anti-tumor drug as it is effective against
both bacterial and eukaryotic cells.
Rifampin binds to R N A p o ly m e r a s e
b. Inhibition by enzyme-binding: inhibits and prevents chain extension
beyond three nucleotides.
RNAP binding and/or enzymatic activity R N A p o ly m e r a s e from eukaryotic

cells does not bind rifampin, and
RNA synthesis is unaffected.
Rifampicin - used to treat mycobacterial

infections (TB, leprosy) (Ferrier7 31.10)
IV. RNA Biosynthesis in Eukaryotes
A. General Features
Shared with Bacteria:

Requires DNA template,
No primer,
5 -> 3 directional synthesis,
limited proofreading
Unique to Eukaryotes:
Compartmentalization: transcription (in the nucleus) is
separated from translation (in the cytosol)
Chromatin Remodeling: DNA wound tightly around histone
proteins must be relaxed in order to be accessible to the
transcription machinery. This is mediated by Histone Acetyl-
Transferases and Histone Deacetylases.
RNA processing: Primary transcripts get modified into
biologically active forms.
RNA Polymerase: There are THREE functionally distinct
polymerases in the eukaryotic nucleus. Organelles such as
the mitochondria and chloroplasts also have RNAPs, but
these more closely resemble bacterial polymerase.
Enzyme Location Product R elative

A ctivity
RNA polymerase 1 nucleolus ribosomal RNA 50-70%

RNA polymerase II nucleoplasm messenger RNA 29-40%
RNA polymerase III nucleoplasm small RNAs (tRNAs) ~ 10%
B. RNA Polymerase II (RNAP-II) & protein encoding mRNAs
NOTE: RNAP-II also synthesizes some small nuclear RNA

(snRNA) as well.
Further, the actual product of RNAP-II for protein encoding genes
is heteronuclear RNA (hnRNA) not mRNA. It doesnt become an
actual mRNA till it is fully processed (see point 3 below on
processing).
1. Gene Structure:
Incredibly heterogeneous since there are many different
genes to make many different proteins!
Core Promoters consist of several elements: TATA box,
Initiator element, and a Downstream promoter element,
(though not all elements are present at all promoters)
TATA box is analogous to the -10 and -35 boxes of
bacteria
__________________________ TATA Inr DPE
-1 0 0 -3 0 +1
The initiator element alone is sufficient to recruit

RNAP-II and initiate transcription at the proper site.
(though it makes a weak promoter on its own)
Mutation of the TATA results in an 80% decrease in (3-
globin mRNA. The resulting disease is (3-thalassemia.
2. E n zy m e : RNAP-II is a 10 - 12 s u b u n it c o m p le x (s o m e s h a r e d
with RNAP-I a n d RNAP-III, s o m e a r e u n iq u e ) th a t e m p lo y s
m a n y a c c e s s o r y fa c to rs.
a. B A SA L tra n sc rip tio n fa c to rs (s a m e a t e v e ry g e n e to b e
tra n s c rib e d )
- TFII- A, B, D, E, F, a n d H
TFII-D: a ls o c a lle d th e TATA binding p ro tein o r T B P -
a bit of a m is n o m e r a s s u b u n its of TFII-D a r e a ls o
involved in binding th e initiator e le m e n t a n d th e
d o w n s tre a m e le m e n t!
n F unction: B in d s t h e s e e le m e n ts , re c ru its th e
tra n sc rip tio n initiation c o m p le x a n d d ire c ts
RNAP-II to th e p ro p e r s ta rt site.
b. R e g u la to ry tra n sc rip tio n fa c to rs (sin c e w e d o n o t w a n t to

tra n s c rib e e v e r y g e n e all th e tim e!)
T h e s e a r e sp e c ific for p a rtic u la r g e n e s (or s e t s of
g en es)
C a n h a v e binding s ite s m u ch fu rth e r from th e initiation
s ite in s e q u e n c e
n R e m e m b e r 3-D s tru c tu re th o u g h ! T h e y m a y b e
q u ite c lo s e in s p a c e
F unction: B in d s t h e s e e le m e n ts a n d e ith e r positively o r
n e g a tiv e ly re g u la te th e initiation of tra n sc rip tio n for
th e s e g en es.
(J. Simmons, MSU)

c. M ushroom Poisoning
W ithin 12-24 hours, ingestion of Amanita phalloides
causes severe abdom inal cramps, vom iting and
diarrhea.
20% of cases are fatal
Toxin 1a-am anitin is a potent inhibitor o f RNAPII
3. Processing: This is w here the prim ary transcript (hnR N A) is

converted into the m ature transcript (m RNA)
C onsists of 3 processing events:
- capping of the 5 end
- polyadenylation of the 3 end
- splicing
a. C a p p in g :
i. Addition of G TP in a 5 -5 triphosphate
linkage
ii. M ethylation of the G base ^ 7-m ethyl-G
iii. M ethylation of the ribose sugar on the 1st
(and som etim es the 2 nd) nucleotide.
iv. Dual Function:
a) Serves as a signal for subsequent
processing by m arking this transcript as
an m RN A
b) Serves as a recognition m arker for
ribosom es - critical fo r efficient translation!
b. P o ly a d e n y la tio n :
i. T erm ination of transcription is NOT precise. RNAP-II goes
on and ju st eventually falls off.
ii. The transcript does, however, need a precise end. This
discrete end is derived from
a) Site specific cleavage: near the end, there is a
Polyadenylation Signal (very precise signal with a
precise sequence - m utation of a single nt m esses up
tail addition) that tells a nuclease to cut ~20 - 30 nt
downstream .
b) Addition of multiple A nucleotides: (not coded for in the
gene and no template required). After the nuclease
cut, Poly A Polymerase adds -200 nt poly A.
iii. Function: stability

a) The role of the poly A tail is to block the binding of
nucleases and allow the toleration of a little
degradation (the loss of -100 nt is OK...) This endows
the mRNA with stability by protecting it from digestion.
b) Exception to the rule: histone mRNAs are not
polyadenylated - since they are only needed during S
phase of replication! Stability is not an issue for them...
* Important Features or Regions of the mature mRNA: (from the left)

1. 5 Cap
2. 5 Untranslated region (UTR)
3. AUG start codon
4. ORF (coding region)
5. STOP codon
6. 3 UTR
7. Poly A tail
The UTRs (sections of the mRNA that do not code for protein) are still
important! For example: In iron metabolism, the Iron Responsive
Elements (IREs) are in the 3 UTR for transferrin and in the 5 UTR for
ferritin. - allow the cell to respond to the local iron levels!
d. Splicing:
i. Some mRNA encoding genes are
multi-partite in that they have
coding regions that are interrupted
by non-coding (or alternately
coding) regions.
a. These regions not being
incorporated into the mature
mRNA are called INTRONS -
intervening sequences - need to
be removed
b. As opposed to EXONS - the
coding sequences that are kept
and joined. (Ferrier7_3.18)
ii. Removal of introns requires precision, (otherwise the

reading frame will be out of register)
iii. Splicing sites are specified by sequences in the hnRNA
5 splice site 3 splice site
(donor site) branch site (acceptor site)
i I
5.....AGlGU ... ..C A G l G ..........
Upstream ^ ___ Downstream
Intron exon
exon
iv. Splicing Machinery (Spliceosome) is analogous to ribosomes

and contains several hundred proteins and several small
nuclear RNAs (snRNA).
Each of the snRNAs is part of a larger complex called a
Small Nuclear Ribonucleoprotein Particle orsnRNP,
(snurp).
Each snRNP contains one snRNA and its own set of
proteins.
There are 5 snRNPs - and each is characterized by a unique snRNA

and has a specific role in the splicing process: snRNPs and other
proteins of the spliceosome carry out the endonuclease cleavage and
ligation reactions.
Medical Significance: snRNPs can be antigens in auto-immune
disorders
(e.g., systemic lupus erythematosis)
iv. multiple introns:
- Ordered splicing
Example: a gene has 3 exons separated by 2 introns
can be spliced in exact order, 1 to 2, 2 to 3,
without skipping (not 1 to 3)
EXON 11EXON 2 | EXON 3).
EXON T]A[ixON2]A[ EXON

OR
EXON EXON 3^
EXON 2
b
EXON 1 1 EXON 3|-
(Ferrier7_31.19)
- Alternative splicing: joining 1 to 3, skipping 2, can give rise

to different polypeptide products (may be a tissue-specific
mechanism to get similar but not identical proteins)
For this example (tropomyosin): One of these products may be in

striated muscle and another in smooth muscle... therefore the Ca2+
sensitivity in one muscle may be slightly different than another!
(Ferrier7_33.12)
M e d ica l S ig n ific a n c e : A berrant splicing can cause disease: p-
thalassem ia
Norm al p-globin m RNA processing:

5-AGAU-------(1307 nt)- A G G U - (635 nt)- C A G G X Y Z ------- (1000 nt)---3 (2957 nt)
5-AGAU------(1307 nt)-----AGGXYZ------(1000 nt)---3 (2317 nt)
M utant p-globin mRNA:
5-A G G U ------ (1307 nt)- A G G U - (635 nt)- C A G G X Y Z -------- (1000 nt)---3 (2957 nt)
5-AGGXYZ------(1000 nt)---3 (1006 nt)
The resulting m utant p-globin m RN A is shorter and likely less stable

The resulting m utant p-globin polypeptide will be shorter; m ay be out of
frame, and likely less stable
NO T Functional!
T here will be an im balance in production of functional p-chain relative to

a-chain p-thalassem ia
4. RNA Transport:
A ll m R N A s - from the nucleus w here it is transcribed and
processed, to the cytoplasm w here it can serve as a tem plate
fo r translation.
REQUIRED READING
IV. Mechanism of Splicing
A. The branch site (usually an A) attacks the 5

splice site along with the rest of the splicing 5* Splice
donor site
3 Splice
acceptor site
machinery and cuts using an endonuclease / \

activity. 5' EXON 1 G pGU ( AJ AG exon i]r
snR N P s
B. This results in a lariat (loop) structure within The primary

transcript
the intron. combines with
snRNPs to form a
spliceosome.
C. Next the AG of the upstream exon joins with

the G in the downstream exon with a ligase.
Adp[ EXON2]3*
D. Product: Two exons joined together with an The 2 -O H of the branch site A
attacks the 5 '-p at the splice
intron spliced out. donor site of the intron, forming

a 2' > 5' phosphodicstcr bond
and a lariat.
IV. RNA Silencing: The process by which small non

coding RNAs act as regulators of gene expression.
There are two kinds of small non-coding RNAs: short
interfering RNA (siRNA) and micro RNA (miRNA). The The free 3 -O H of
exon 1 then attacks
difference between siRNA and miRNA is based the 5 - p at the splice

acceptor site,
forming a phospho-
primarily on their source. diester bond that
joins exons 1 and 2.
A. Source
a. siRNA - short interfering RNA Excised intron
Source: exogenous - from a foreign ("lariat")
5' [EXON1JÊXON2J3'
challenge such as a virus. Mature m RNA
Viruses can have RNA or DNA genomes. (Femer7_3i.i8)

In this figure you will see how a siRNA can be generated from
either a DNA virus (left) or RNA virus (right).
Transtribed RNA from Transcribed RNA from
a DMA virus genome a RNA virus genome
RNA strand 1
f >
d sD N A
Complementary RNA
RNA strand 2
dsRNA Processed by DICER enzyme complex

^ into ~21 nt pieces -> siRNA
siRNA --------- ---------
b. miRNA - micro RNA

Source: endogenous - from the organisms genome. Micro
RNA genes are plentiful and can be found throughout the
genome including within some introns of protein encoding
genes! (termed mirtrons)
Transcribed from endogenous genome

Must be self-complementary!
RNA
dsDNA
RNA folds into This secondary structure is processed

a "hairpin" by DICER enzyme complex again into
~21 nt pieces -> miRNA
B. Mechanism
One strand of the 18-24 nt RNA is loaded into the RISC. (RNA
Induced Silencing Complex). This guide strand targets the
complex to an appropriate mRNA and binds to its target region
(usually in the 3 UTR).
C. Outcomes - The overall outcome for both mechanisms is the

same: a lower level of protein expression for the targeted gene.
Hence, this process is called a gene knock down (NOT a knock
out)
a. Translational interference - Since the complex is bound to the
mRNA, translation by ribosomes is physically blocked,
(sometimes reversible!)
Ribosomes cannot translate through the blockage!

b. mRNA degredation - binding may also mark the mRNA for

nuclease cleavage and degredation
Nuclease cleavage begins the process of mRNA degredation.

D. Biological Context
a. It is estimated that ~1/3 of all genes are regulated by miRNA!
b. Embryology: active miRNAs are involved in maintaining stem
cell pluripotency and in determining cell fate upon
differentiation. It is also involved in mammalian neurogenesis
and certain miRNAs appear to be expressed in waves during
development.
c. Provides a natural defense against viruses and related
challenges
E. Research and Medical Applications

a. Targeted down-regulation of specific genes in specific tissues
b. Determining the existing miRNA profiles in certain normal and
diseased/cancerous tissues can aid in the determination of
cancers developmental lineage: tissue of origin and
differentiation stage.
c. Altering miRNA profiles can be immunogenic in certain tissues
d. Provides an engineered defense against viral infections such as
Ebola, HIV, Hepatitis B & C, HSV2
i. Some are still in animal model research, others are
already in clinical trials!
e. Provides possible treatment for neurological disorders such as
Parkinsons, Alzheimers, ALS, etc...
i. Some have completed non-human primate trials and are
awaiting the start of their clinical trials!
Summary Table of RNA types, features, and functions:

This is not meant to be comprehensive, but a study guide
Type Designation Physical Features Function
Heteronuclear hnRNA Direct unprocessed transcript of RNAPII. Protein encoding
Contains exons & introns. Made and
processed in nucleus.
Messenger mRNA Processed from hnRNA. Low abundance, Protein encoding
high diversity, variable size. Contains
5cap, 3 tail, and only exons within its 1
open reading frame.
Ribosomal rRNA High abundance, low diversity, size Structural & enzymatic
measured in units of Svedberg (S). Made component of ribosomes
and assembled in nucleolus.
Usually transcribed by RNAPI
Transfer tRNA Intermediate abundance and diversity, decode message: match
uniform in size and shape. codon w/ a.a.
Transcript of RNAPIII
Small nuclear snRNA 1 snRNA + many proteins = Enzymatic component
snRibonucleoprotein particle (snRNP). of splicing machinery
Made and function in nucleus.
Transcript of RNAPIII
Short siRNA Double stranded RNA of ~21 nt processed Gene silencing- reduce
interfering from either DNA or RNA of an exogenous expression of genes.
source
Micro- miRNA Double stranded RNA of ~21 nt processed Gene silencing- reduce
interfering from an endogenous transcript of a self- expression of genes.
complementary region of the genome. Source-endogenous (may
be reversible)
Guide gRNA 1 gRNA + proteins = editosome. RNA editing-increases
Complementary to portion of mRNA to be diversity of proteome
edited.
V. RNA Editing: Process by which a fully processed mRNA can be edited
to give rise to protein variation. This is another method of achieving
diversity from our genome!
Example: Apolipoproteins B-100 and B-48 come from the same mRNA!
However, the mRNA in the small intestine is edited to create a shorter
protein (named B-48 since it is 48% of the original length). The deaminase
is not expressed in the liver leaving the mRNA to be translated into the full
length protein; hence B-100 (100% of the length)
Same gene, Same mRNA, Two different protein products!
Lipoprotein assembly LDL receptor binding
-C O O 4536 aa
Apo B-100
Translation
5'-| CAA 1-3'

mRNA
I RNA editing by
NH4+-J deam ination
5'-|_____ UAA 1-3'

Edited mRNA
Translation
H,N+ l-COO 2152 aa
Apo B-48
(J. Simmons, MSU)
VI. Catalytic RNA: Often termed Ribozymes, refers to enzyme activity

in an RNA.
A. Self Splicing RNA

Example: ribosomal RNA from Tetrahymena (a protozoan)
splicing both "endonuclease" (cut) and "ligase" Qoin)
activities accomplished by the RNA itself no protein is
necessary!
Intron Intron
GTP
Exon 1 Exon 2
/
EndonucleaseCleavage
NOT Technically a catalyst! There is no self-renewal.

(J. Simmons, MSU)
B. T ransfer RNA Processing

Example: ribonuclease P from E. coli
cuts precursor m olecule precisely to form correct 5' and 3 ends
o f tR N A s
Native enzym e has RNA and protein com ponents
However, the RNA com ponent alone is sufficient to correctly and efficiently
cleave the precursor!
(J. Simmons, MSU)
C. A dditional catalytic RNAs:

a. Spliceosom e: the snR N As do the cutting and ligation!
b. Ribosom e: rRN A is the catalyst fo r peptide bond form ation!
i. i.e. the peptidyl transfer reaction.
Protein Translation
In this lecture, we will examine the details of the process of protein biosynthesis, using E. coli as our
model. We will see how the various classes of RNAs are prepared for their roles in translation. We will
then focus on the details of initiation, elongation, and termination of translation. Inhibitors of translation,
frequently used as antibiotics, will be briefly described. We will then review the process of translation in
eukaryotes, noting critical similarities and differences to the bacterial model.
Suggested Reading:
Ferrier: Chapter 32: Protein Synthesis - from Overview through "Steps in Translation
Turnpenny and Ellard: Chapter 2: The Cellular and Molecular Basis of Inheritance - Translation -
through "Transfer RNA": and "The Genetic Code (p. 19-20)
Ferrier 6th ed [available online through the online MSU library resource LWW Health Library
(http://meded.lwwhealthlibrarv.com.13roxvl.cl.msu.edu/books.as13x) look under: video and audio:
Animation: Protein Synthesis (there are two options: Overview or The Details)
Objectives:
1. Describe the components of ribosomes and their role in protein translation and distinguish the
subunits of prokaryotic versus eukaryotic ribosomes.
2. Delineate the non-ribosomal components of the protein synthesis machinery (codons on mRNA, anti
codons on tRNA) and the role of amino-acyl tRNA synthetase.
3. Describe the process by which the mRNA code is translated into a protein sequence.
4. Identify the key differences between prokaryotic versus eukaryotic translation (recognition of
initiation codon; first amino acid incorporated; transcription-translation coupling).
5. Recognize how certain antibiotics function by inhibiting different steps of protein synthesis.
6. Understand the basis of protein folding.
I. Translation of Genetic Information

1. Describe the roles of mRNA, tRNA, rRNA, codons, and anticodons in the translation of
genetic information from nucleic acid sequence to polypeptide sequence. (O b j. 1 a n d 2 )
2. Given a table of the genetic code but ignoring the complications of the Wobble Hypothesis,
apply the rules of genetic coding to the following situations: (O b j. 3 )
A. Given a codon, identify the corresponding anticodon(s) and amino acid.
B. Given an anticodon, identify the corresponding codon(s) and amino acid.
C. Given an amino acid, identify the corresponding codon(s) and anticodon(s).
II. Translational Machinery

1. Describe the ribosomes of bacteria and eukaryotes, including their size, subunits, and the
RNAs and approximate protein composition within each subunit. (O b j. 1)
2. Describe the process of amino acid activation, including: (O b j. 2 a n d O b j. 3)
A. The role and specificity of amino acyl-tRNA synthetases.
B. The position of the covalent bond formed with respect to both amino acid and tRNA.
C. The role of ATP and the "cost" in high-energy bonds.
III. Translation Mechanism
1. Compare and contrast the mechanisms used in E. coli and eukaryotes for identifying start
codons. (Obj. 1, Obj. 3 and Obj. 4)
2. Describe the sequence of events leading to a translation initiation complex, including small
and large subunits, mRNA, initiator tRNA, GTP, and (unspecified) initiation factors. (Obj. 1,
Obj. 3 and Obj. 4)
3. Summarize the three principal steps in elongation, including the molecules that occupy the P
and A sites within the ribosome and the role of GTP. Describe the formation of peptide bonds,
including the implication for the direction of polypeptide chain growth. (Obj. 3)
4. Sketch or recognize and label a diagram representing polysomes. (Obj. 3)
5. Sketch or recognize and label a diagram representing the coupling of transcription and
translation in bacteria. (Obj. 1, Obj. 3 and Obj. 4)
6. Briefly describe the mechanism by which stop codons are recognized and translation is
terminated. (Obj. 3)
7. Recognize or recall the steps in translation that may be sensitive to effects of antibiotics
(actions of specific antibiotics need not be memorized at this time). (Obj. 1, Obj. 3, Obj. 4 and
Obj. 5)
IV. Higher-order Protein Structure

1. Describe the forces that drive polypeptides to fold into specific secondary and tertiary
structures. (Review) (Obj. 6)
2. Define chaperonins and describe their function. (Obj. 6)
I. Translation of Genetic Information: Protein Synthesis
DNA RNA Protein This last step is our focus for tins section
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I I I
TRANSCRIPTION
JL
tRNA
5' UTR Protein-coding region UTR 3'
m RNA
I
TRANSLATION
N -term inus C -term inus
Protein
A. mRNA carries the information (sequence of nucleotides) to be translated into a sequence of

amino acids.
The whole mRNA is critical, but the only part to be directly translated, is the Open Reading
Frame!
UAA
UAG
AUG UGA
C. Codons of the Genetic Code are Unambiguous and Degenerate (or redundant) meaning that while
each codon codes for only one amino acid, a single amino acid may be encoded by more than one
codon.
First Third
position
There are 64 positions on the Codon Table: position
(5' end) U
Second position
C A G (3' end)
(4x4x4) with A,C,G,U as options at each position Phe Ser Tyr Cys U
61 codons and 3 STOP codons Phe Ser Tyr Cys C
Leu Ser Stop Stop A
However, there are only 20 amino acids! U Leu Ser Stop Trp G
Leu Pro His Arg U
Leu Pro His Arg C
This implies several things: Leu Pro Gin Arg A
C Leu Pro Gin Arg G
1. Iso-accepting tRNAs: different tRNAs may carry the lie

lie
Thr
Thr
Asn
Asn
Ser
Ser
U
C
same amino acid lie Thr Lys Arg A
2. "Wobble Hypothesis: Some tRNAs can read more than A M et Thr Lys Arg G
Val Ala Asp Gly U
one codon (or one anti-codon can base pair with more Val Ala Asp Gly C
than one codon) Val Ala Glu Gly A
G Val Ala Glu Gly G
Exercise to confirm our understanding:
Exercise 1: For each of the following amino acids, identify the codons and anticodons.
Amino Acids Met Glu Ala

Codons:
Anticodons:
Exercise 2: For each of the following anticodons, identify the corresponding codon(s) and amino acid.
Anticodon 3 -ACC-5 3-GUG-5

Codon(s):
Amino Acid:
Rules of Wobble:
Codons are typically written 5'-3' and anti-codons are typically written 3-5'
The 3' position of the codon (or 5 of the anti-codon) is the wobble position
G=U base pairs are allowed (as in other RNA)
Anti-codons can contain Inosine in the wobble position which allows I=U, I=C, and I=A
base pairs.
Anticodon Anticodon
3' 5 3' _v
A A G A A G
U U U U U C
5' 3 5 ' 3'
Codon Codon
Anticodon Anticodon Anticodon
3'
C G I
y 3' 5r
C G I
3' 5'
C G I
v J 5 h v
Traditional base Nontraditional
pairing observed base-pairing is
G C U G C C G C A in first and second possible between
positions of codon: the third (3)
5 3 5 3' 5 3 tRNA mRNA position of the
codon and the
first (5) position
Codon Codon Codon of the anticodon:
tRNA mRNA
A u
NOTE: The codons to be recognized by any given tRNA must S)
G
all code for the same ammo acid or the code would be
&
ambiguous! ... and that would be very bad. c G
u
1 c
A
tRNA anticodons are also Unambiguous!
The "Wobble" Rules of
Codon-Anticodon Pairing
5' Nucleotide 3 Nucleotide

of Anticodon of Codon
C G
A U
U A or G
G C o rU
1 U, C, or A
Inosine Adenine
3' Nucleotide 5' Nucleotide
of Codon of Anticodon
G C ,U
A I, u
C l,G
U 1, G, A
II. Translational Machinery: the Ribosomes
A. Structure: rRNA is a cntical part of the ribosome, but it is not the only part. Shared and specific
proteins are employed in the composition of the nbosomal subunits.
Eukaryotic
Bacterial Eukaryotic Ribosome
1. Subunits Small 30S 40S
Large 50S 60S
(small+large) Ribosome 70S 80S
2. Components
5S 5S, 5.8S
large
rRNAs 16S 18S s u b u n it
23 S 28S
Proteins MANY MANY
SSU proteins LSU

p ro te in s
(S. Triezenberg and J. Wang, MSU) 1 8S rR NA

5S rR NA 28S rRNA
B. Function: This machinery of translation is responsible for holding the mRNA and the tRNA
together and for catalyzing the formation of the peptide bonds.
C. tRNAs and Amino Acid activation: Each tRNA must have the proper amino acid attached in
preparation to be used in protein synthesis. This process must be specific and accurate to
maintain the integrity of the genetic code.
Activate amino acid for peptide bond formation
Attach amino acid to the proper tRNA to create an aminoacyl-tRNA
l. Enzyme: Aminoacyl-tRNA Synthetase: 2 step reaction NH}

/
a. There is a distinct aminoacyl-tRNA synthetase for R c *
every amino acid V

b. Enzyme has proofreading capability - to make o- pp,
sure it gets the right aa attached to the tRNA. J
2 Pi
2. Energy: provided by ATP (2 high energy bonds for
every amino acid activated!)
3. Product: step 1-aminoacyl-AMP (activated)

step 2 - "charged aminoacyl-tRNA (S. Triezenberg and J.
What is a charged tRNA ?

The bond between the AMP and the amino acid stores energy. Since every tRNA 3 end is processed to
end in 5-CCA-3'. when the tRNA displaces the AMP on the aminoacyl-AMP, it will essentially be
replacing one adenine nucleotide for another - so the energy is preserved! It is this stored energy that will
be used to make the peptide bond.
Proofreading Example: Thr-tRNA Synthetase will CLEAVE a mischarged Ser-tRNAxhr, but will leave
a correctly charged Thr-tRNAxhr alone.
Therefore, the enzyme chooses both the tRNA (based on the anticodon) and the amino acid.
rNote: Although the proofreading mechanism of the aminoacyl-tRNA synthetase is very accurate,
mistakes may occur, and a tRNA may be mischarged (e.g. Ala-tRNAThr)].
III. Translation Mechanism (Start Continue STOP)
A. Initiation (Start): The starting amino acid is always Methionine which is coded by 5-AUG-3
However, the amino acid met occurs at other positions of a polypeptide, NOT just #1, so how
does the translational machinery know which AUG to use for a start codon?
Open reading
frame
Start Stop
codon codon (S. Triezenberg and J. Wang, MSU)
Also remember that bacteria have polycistronic mRNA, so there can be more than one AUG
that should be used as a start codon!
The Solution in bacteria is the use of a Ribosomal Recognition Site (RRS): a consensus
sequence that is recognized and bound by ribosomes allowing the next encountered AUG
codon to be used as a start codon.
Sequence on the mRNA that base pairs with the 16S rRNA (small subunit of bacterial
ribosome) - this is how the ribosome assembles! The small subunit binds the RRS, recruits the
large subunit, then finds amino acid #1 (first AUG codon)!
The Ribosomal Recognition Site is actually what allows bacterial mRNAs to be polycistronic!
Each ORF can be recognized by its own ribosomal recognition site. The RRS also serves to
distinguish AUG for Start versus an AUG for an internal Met in each ORF.
ORF 1 ORF 2
... G G C A A U U C A G G A G G U G C U U C U 4U G G A ...
ribosome
recognition site
The Solution in Eukaryotes is the scanning model: The 5 cap structure is the RRS in
eukaryotes. The 40S ribosomal subunit binds to the 5 end and scans downstream to find the first
AUG and uses that to start.
Ribosome
binding Open reading
frame '
r AUGGCUNNNNNNNNNNNNNNNNNNNNNNNNUAG
------------ - AAAAAAfnr
................................................................................................
Eukaryotic mRNA can only be monocistronic (one ORF); only one start AUG (the first one from
the 5 end). Proper capping at the 5 end is critical, otherwise, there are greatly reduced initiation
rates (but never the less, they can undergo translation)
Mechanism of Initiation:
Preinitiation complex 70S initiation complex

GTP GDP

1. Small subunit recognizes the RRS and binds.
2. Large subunit is recruited and locked into place using the energy of one GTP
3. Assembled ribosome has 2 sites: a P-site containing the met-aminoacyl-tRNA and an A-site
which is empty. Note the polarity of the tRNA.
P-site: is the Peptidyl site or the site where the growing peptide chain will be
A-site: is the acceptor site or where the incoming aminoacyl-tRNA will bind
(there is also evidence for an E-site or Exit site where empty tRNA sits before leaving, but we
wont worry about it here...)
B. Elongation
A three step mechanism:
70s initiation complex Delivery of
(S. Triezenberg and J. W ang, MSU)
Mechanism:
The P-site contains the first aminoacyl-tRNA and the A-site begins empty.
The next aminoacyl-tRNA to be brought in is determined by the next codon on the
mRNA.
o Loading the aa-tRNA uses the energy of one GTP.
The amino end of the 2ndamino acid attacks the carboxy end of the 1st, releasing it from its
tRNA carrier and creating a peptidyl-tRNA in the A-site.
o This reaction uses the energy stored in the aa-tRNA bond and is catalyzed by the
rRNA of the ribosome.
Once complete, the ribosome slides down the mRNA one codon placing the peptidyl-
tRNA in the P-site and again leaving the A-site empty.
o This translocation requires the energy of another molecule of GTP.
This process repeats for every codon of the ORF.
INote: Ribosomes do NOT have proofreading activity. The codon on the mRNA is what determines the
next aminoacyl-tRNA to be incorporated during elongation. In the event a mischarged tRNA (e.g. Ala-
tRNAThr) is NOT caught by the proofreading mechanism of the aminoacyl-tRNA synthetase this
mischarged amino acid (Ala in this case) will be incorporated into the protein being formed following the
anti-codon of the incorrectly charged aminoacyl-tRNA (Thr anti-codon)].
Chemical Details: the peptide chain grows from amino terminus to carboxy terminus (N -> C)
as the mRNA is read in a 5' -> 3' direction
The amino groups of the aa-tRNAs are free while the carboxy groups are busy in bonds. The
amino group of the first Met remains free and becomes the amino terminus of the polypeptide.

Polysomes: (orpolyribosomes) Many ribosomes can translate a given mRNA simultaneously.

Coupled Transcription and Translation: in bacteria only!
Allows translation to begin before transcription is complete, because there is no nucleus v.
cytoplasm to separate the two processes.
Impossible in eukaryotes1

C. Termination
1. Stop codons (UAA, UAG, UGA) - do not code for an amino acid
2. There are NO stop tRNAs to recognize the stop codons
3. Termination factors are proteins that bind to the stop codons and aid in the release of the
new polypeptide and dissociation of the ribosome.
4. Dissociation of the Ribosome - when ribosomes arrive at the STOP codon, it senses that
there is no tRNA to come into the A-site. Termination factors cleave the peptide from the
last aa-tRNA and uses the energy of one last GTP to dissociate the ribosome
Cell Factor Function

There are protein factors that are used in the three stages of
Initiation
translation.
P IF-2-GTP Bring charged initiat-
E elF-2-GTP ing tRNA to P site
Clinical Relevance: these protein factors are the target of
P IF-3 Prevent association
antibiotics as well as bacterial toxins (diphtheria and pseudomonas
E elF-3 of subunits toxins) during inhibition of eukaryotic translation. These factors
Elongation
are also used by the cell as a way of regulating translation [e.g.
Bring all other
interferon inhibits translation in virally infected cells by
P EF-Tu-GTP phosphorylating eIF-2 (phosphorylated eIF-2 is inactive)].
E
charged tRNAs to
EF1-GTP
A site
P EF-Ts Guanine nucleotide
E E F -IP y exchange factor
P EF-G-GTP
E
Translocation
EF-2-GTP
Term ination
P RF-1,2 Recognize "stop"
E eRF codons
P RF-3-GTP
E
Release of other RFs
eRF-3-GTP
(Ferrier7 F32.14 p458)
D. Inhibitors of Translation
Antibiotic inhibitors of protein synthesis
Antibiotic Action
Streptomycin Inhibits initiation and causes misreading of mRNA

Neomycin Binds 30S subunit, affects initiation, causes misreading
Tetracycline Binds to the 30S subunit and inhibits binding of aminoacyl-tRNAs
Chloramphenicol Inhibits the peptidyl transferase activity of the 50S ribosomal subunit
Erythromycin Binds to the 50S subunit and inhibits translocation
Puromycin Causes premature chain termination by acting as an analog of aminoacyl-
tRNA (procaryotes and eukaryotes)
Cycloheximide Inhibits peptidyltransferase reaction of 60S subunit
Diphtheria toxin Covalently modifies an elongation factor by ADP-ribosylation.
Enzvmatic: drastic effect!
Note: Antibiotics can inhibit at different steps in the translation process:

Initiation for streptomycin vs. Elongation for Chloramphenicol
Antibiotics can also be specific for either bacteria, eukaryotes, or affect both!
IV. Higher-order Protein Structure
The primary structure (amino acid sequence) of a protein determines all subsequent higher order
structures: secondary structure (a-helix, p-sheet), tertiary structure (globular fold), and quaternary
structure (association of domains or subunits) and all of this is driven by entropy.
Chciperonins are a set (or family) of proteins that help other proteins fold properly.
e.g. they may shield certain regions from water until translation completes so the proper
hydrophobic interactions can form.
(Lieberman4_F7.17)

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Carbohydrate Metabolism

Measurable Outcomes: You should be able to ...

A. Describe the structural features of mitochondria. (Obj. 1)

(J. LaPres and C. Wilkins, MSU)

T o g e t m a x im u m A T P fro m g lu c o s e (38 to ta l),

Figure 3.1, p. 66: Basic structure of a mitochondrion

C. Intermembrane space (a.k.a. inner membrane space)

2. Has high concentration o f________________

3. Contains 10+ copies of th e ________________________

F. Functional Roles of the mitochondria. (Figure 3.2, p. 67)

2. M ultienzym e com plex (is huge): has 5 different enzym es--

5. A c lin ic a l n o te : The lipoam ide cofactor of E 2 is the site o f inhibition

2. P hosphatase: C leaves off t h e _______________ , w h ic h ____

G. PDH com plex is also in h ib ite d by:

H. This bridging step is a ke y re g u la to ry p o in t:

2. C onversion o f the glucose carbons to acetyl C oA com m its them to

3. Thus, PDH com plex activity controls the fate of pyruvate:

Questions relating to case:

1. Why were the plasma concentration of pyruvate, lactate, and alanine

2. Enzyme activity of the PDH complex, a-ketoglutarate dehydrogenase

3. Lipoic acid added to the fibroblast homogenates did not stimulate

5. Restriction of dietary carbohydrate to 40% of the food calories has been

Skin fibroblasts in the diagnosis of genetic diseases

1. High plasma concentrations

5. Dietary carbohydrate and acidosis

Matalon R et ai: Lipoamide dehydrogenase deficiency with primary lactic acidosis:

Mituda S et ai: pyruvate dehydrogenase subcomplex with lipoamide dehydrogenase

THE TRICARBOXYLIC ACID (TCA) CYCLE

Measurable Outcomes: You should be able to ...

F. Define the terms: amphibolic and anaplerotic. (Obj. 1 and 2)

D. F ig u re 3.5, p. 73: The n e t reaction o f the TC A cycle:

F ig u re 3.6, p. 75: The TC A cycle

2. The hydrolysis o f_____________pulls the overall reaction far in the

3. Citrate can also be used as the transporter of:

STEP 2Isomerization of citrate to isocitrate. (Figure 3.8, p. 77)

2. The isomerization involves two steps both catalyzed by

4. Enzyme named after__________________.

2 . ____________________ catalyzes both the oxid/red reaction and

3. The intermediate_______________is a n ___________________ .

4 . _______________ is an amino acid precursor; can be converted to

STEP 4The second redox reaction of the cycle. Shown as a simplified

STEP 7A hydration step to go from an alkene to a secondary alcohol.

1. Citrate synthase (step 1)

2. Other regulated enzymes of the TCA cycle

G. TCA Cycle as a Provider of Intermediates for Biosynthetic Pathways

2. Problem: TCA cycle intermediates must be replenished if any

1 ATP (hydrolysis) = -7.3 kcal/mol

If you were to completely oxidize glucose = -686 kcal/mol

ELECTRON TRANSPORT CHAIN AND

Measurable Outcomes: You should be able to ...

Note: Ratio of 3 ATP made for 1 NADH consumed.

2. This_________________ is used to drive the synthesis of ATP

F. Overview of the Electron Transport Chain

H. How does the electron transport chain work?

____________________ : Which suggests that the mitochondria

I. General Structure of the ETC

2. Complex V: a n ___________membrane protein complex that

Figure 4.3, p. 90, The electron transport chain

Figure 4.4, p. 91: FMN and FMNH2

b. Coenzyme Q (ubiquinone) is reduced to ubiquinol (C0 QH2 )

Figure 4.5, p. 92: Oxidized and reduced structures of CoQ.

J. The prosthetic groups of the ETC2 major categories (continued ):

Cytochromes c: is soluble (carries e- between complexes III

2. P hosphatase: C leaves off t h e ___________ , w h ic h

3. The intermediate_is a n _____ .