1

Essential
Biochemistry

Third Edition
Charlotte W. Pratt | Kathleen Cornely

Lecture Notes for

Chapter 19
Regulation of Mammalian Fuel Metabolism

+Effects of Glucagon

glucagon
Glucagon

is small (29 residue) peptide

hormone

Synthesized and released by pancreatic

cells

Produced

in response to drop in blood

glucose levels

Insulin high blood glucose

Stimulates

production of glucose by liver

Gluconeogenosis (glucose from scratch)

Glycogenolysis (stored glucose)

+Effects of Glucagon
Stimulates

production of glucose by liver

Gluconeogenosis (glucose from scratch)

Glycogenolysis (stored glucose)

Stimulates

lipolysis in adipose tissue

Mobilize stored TAGs

Muscle

cells respond to different

hormone

Epinephrine
Stimulates glycogenolysis

glucagon

+Trigger for Fuel

Metabolism

+
Effects of Signaling Cascades
cascade greatly
amplifies effects of
hormones

cAMP

(Liver cell)

amount of hormone
Large effect

Small

Need

to turn off quickly

protein has inherent GTPase

activity
Converts G protein to inactive
GDP-bound form
10,000

Essential Biochemistry
Third Edition
Charlotte W. Pratt | Kathleen Cornely

Lecture Notes for

Chapter 3
From Genes to Proteins

+
Structure of Nucleotides
Nucleic

acids are polymers of

nucleotides

Identity

base

of nucleotide determined by
A and G resemble purine

+
Structure of Nucleotides
Identity

of nucleotide determined by

base

C and T resemble pyrimidine

You dont have to know the structures
but should be able to recognize
purine versus pyrimidine

+
Structure of Nucleotides
RNA

has one different base

DNA

Bases

RNA

are linked to a 5-C sugar

Prime indicates
atom in sugar
rather than base

+
Structure of Nucleotides

10

Nucleosides

become nucleotides when

phosphate groups are present

CTP

is an NTP

oxy

dCTP

is a dNTP

deoxy
CTP is a nucleoside
triphosphate

Both GDP and

CTP are
nucleotides

GDP is a nucleoside
diphosphate

+
Structure of Nucleotides

Some

nucleotides
arent part of DNA

11

+
Structure of DNA
DNA

is a polymer of nucleotides with

phosphodiester bonds

It

R-O-PO2O-R

has directionality

5 phosphate end
3 hydroxyl end
Synthesized 5 to 3
Convention is to read sequence from 5 to 3

12

+Structure of DNA
Canonical

base pairs: purine pairs with

pyrimidine

This

means all Watson-Crick base pairs

have the same dimensions (~11 )

2 H-bonds

3 H-bonds

13

+Structure of DNA

14

This

means the 2 DNA strands are a constant

distance apart

Theres an uneven
twist to the DNA

+Chargaffs rule
gives a clue to base pairing
Chargaffs

Base

rule: the amount of A+G = C+T.

pairs contain a purine and a pyrimidine.

base pairs with T or U; G base pairs with C.

15

+Structure of DNA

16

DNA strands are antiparallel

5

to 3 pairs with 3 to 5
Why?
In order to form H bonds

Sugar-phosphate

backbones are solventexposed

Hydrophilic

http://open.jorum.ac.uk/xmlui/bitstream/handle/12
3456789/956/Items/S377_1_section5.html

+Structure of DNA

17

Base

pairs are
perpendicular to
backbone
Bases

are mostly
hydrophobic
Need to minimize solvent
exposure
Base pairs stack closer
together

http://open.jorum.ac.uk/xmlui/bitstream/handle/12
3456789/956/Items/S377_1_section5.html

+
DNA Base Stacking
Pi

orbitals overlap in
benzene
Alternating

single and
double bonds
Pi conjugation
Very stable electrons (e-)
have more freedom of
movement

18

+
DNA Base Stacking
Bases

in DNA also have

alternating single and double
bonds
They

also have pi orbitals

Pi orbitals of adjacent base pairs
can overlap
Greater freedom of movement
for e-

19

+Structure of DNA
Base

pairs are
perpendicular to
backbone
Movement

of bases
together tilts the backbone
by 30

20

+Structure of DNA
Creates

21

an uneven

twist
Major

groove vs. minor

groove
These are spaces
between backbones
Strands not directly
opposite each other

B-DNA

+
Stability of DNA
Stability

of DNA due to double-stranded

helix
Has

most to do with base-stacking

interactions and hydrophobicity
van der Waals interactions!
Cumulative effect

22

+
Stability of DNA
Stability

23

of DNA due to double-stranded

helix
H-bonding

of bases also a factor

Has more to do with specificity
Ionic interactions
Cations (e.g. Mg2+, Na+, K+)

Base pairs dont

align vertically

View down helical axis of DNA

+
Terminology
DNA

size expressed as bp (base pairs) or as

kb (kilobase pairs)

Oligonucleotide

or oligo = short ssDNA

molecule
DNA

synthesized by polymerases

DNA

digested by nucleases

Exonuclease digests from the end (exo =

outside)
Endonuclease cuts at specific site (endo =
within)

24

+Structure of RNA
RNA
Not

is mostly single stranded

2 separate strands wound

around each other
Not able to form typical helix of BDNA

25

+Structure of RNA

26

Can

form secondary structure by

intramolecular base-pairing
Same

rules of complementarity apply

tRNA

rRNA of small
ribosome
http://rna.ucsc.edu/rnacenter/images/figs/
thermus_16s_2ndry.jpg

27

Essential
Biochemistry

Third Edition
Charlotte W. Pratt | Kathleen Cornely

Lecture Notes for

Chapter 20
DNA Replication and Repair

28

DNA contains cell information

Complete

cell DNA sequence = genome

Genome

of bacteria usually circular

Can be linear

Bacterial
Human
1000x

genome = 0.6 - 9.4 million bp

genome = 4 billion bp

as large as E. coli
But 8x genes: 30,000 (us) vs. 4,000 (E. coli).
E. coli genes use less DNA sequence
About 1000 bases in typical bacterial gene
3000 to 2.4 million in human gene

+Nucleic acids structure

(Deoxy)Ribose

A sugar

Phosphate groups on 5
carbon

5 phosphate of one
nucleotide links to 3 OH of
previous nucleotide

Base

attached to sugar

29

30

Nucleic acid
Information
Information

in the
order of bases
DNA:
Adenine,

Guanine,
Cytosine, Thymine
RNA:
Adenine, Guanine,
Cytosine, Uracil

+
B-type DNA Helix
DNA

31

not a perfectly linear helix

Contains

Major and Minor grooves

Gives regulatory proteins access
to internal bases
Must be able to recognize
sequence
A form: dehydrated form
B form: most common
Z form: unstable;
involved in Transcription?

+DNA Coiling

32

DNA

double helix is a righthanded helix

Thumb points from bottom to
top
Strands coil in the same
direction as the fingers

DNA Tower in Perth, Australia

+DNA Packing

33

Nucleoid of E. coli
Circle of dsDNA 1500x the
size of the cell

Multiple

loops held by anchoring

proteins
Each

loop has coiled DNA

+Twist and Writhe

Twist

(T)

(W)

# of times double helix crosses over itself

Supercoiling

Linking

number (L)

L=T+W

Represents amount of tension in

molecule

Separation of DNA strands

supercoiling in advance of separation

Number of helical turns of one strand

around the other

Writhe

34

Important consideration in DNA

replication

+Supercoiling Compacts DNA

Unsupercoiled
Positive

35

DNA = 1 twist for ~10 bases

supercoils

Supercoils

in left-handed direction
Winding more frequent
Overwinding
Harder to unwind

Rotate top arrow to

align with bottom

+Supercoiling Compacts DNA

Negative

supercoils

Supercoils

in right-handed direction
Winding less frequent
Underwinding
Supercoils

twist DNA

36

+Topoisomerases & Supercoiling

Type

I Topoisomerases

Relieve torsional stress caused by supercoils

Alter Twist

Make single-strand break

Work on both (+) and (-) supercoiling

Energy released

37

+Topoisomerases & Supercoiling

Type

II Topoisomerases

Make

double-strand break
Requires energy
ATP hydrolysis
Can relieve both (+) and (-)
supercoiling
Alter Writhe
DNA gyrase (prokaryotes)
Introduces more (-) supercoiling
Eukaryotes maintain (-)
supercoiling by wrapping DNA
around nucleosomes

38

+Topoisomerases & Supercoiling

Archaeal

topoisomerases

Increases

linking number
Results in positive supercoils
Harder to unwind
More resistant to stress

39

+DNA replication
Semiconservative
Semi

40

replication

Copies

information from one strand to a new

complementary strand
Melt

double-stranded DNA
Polymerize new strand complementary to each
melted single-strand

+Replication Fork
Where

parental strands separate and new

strands synthesized

Begins

at one Ori site in bacteria

Begins

at numerous sites in eukaryotic

cells

41

+Replication Models
Locomotive

model of replication

Replication

machinery moves along DNA like

train on tracks

Factory

model of replication

Replication

machinery is stationary and

feeds DNA through
This is the true model

Fluorescent
foci:
stationary
DNA
synthesis

42

+ DNA Replication
DNA

polymerase synthesizes 5 to 3

Reads

3 to 5
3 OH of last nt attacks P of incoming nt
Breaks phosphoanhydride bond of dNTP; forms
phosphodiester bond in DNA
G0
Irreversible due to hydrolysis of PPi
Must

have 3-OH to add onto

Has proofreading ability

43

+
Obstacles to DNA Polymerase
Can

only extend existing chain

Needs

3-OH to build on

Solution
Use

RNA polymerase (DNA primase) to make

RNA primer
Removed later (no proofreading)

44

+
Obstacles to DNA Polymerase
DNA

45

is antiparallel

No

problem on strand that

runs 3 to 5
Problem encountered with
strand that is 5 to 3
Solution
Synthesize

leading strand
continuously
Synthesize lagging strand
discontinously
Form Okazaki fragments
(100-200 nt in humans)

Synthesis in this way would

pull strands in opposite
directions

+A Model for DNA Replication

46

47

DNA Replication

Helicase

1) Relax supercoiling by nicking one strand

Topoisomerase

2) Unwind helix (separate 2 strands)

Helicase

3) Need proteins to protect ssDNA

SSB: single-strand binding protein

4) RNA primers synthesized

5) DNA replicated
6) RNA removed & replaced with DNA
7) Gaps sealed
8) Supercoiling added
Eukaryotic SSB

+ Sliding
E. coli

Clamp

48

Human