GENE

EXPRESSION
Dolores V. Viliran, MD
Department of Biochemistry & Nutrition
FEU-NRMF,Institute of Medicine
INTRODUCTION

5’ 3’

G~C
A~T 3.4 nm

3’ 5’

Hydrogen bonds
INTRODUCTION

DNA

reverse replication
transcription
DNA

RNA protein
translation

Central Dogma of Molecular Genetics

 SCHEMA OF

GENE EXPRESSION
Replication
(DNA Synthesis)
 Four Phases Of The Eukaryotic Cell Cycle

 M (mitotic) phase – cell division occur

 G1 (gap) phase – longest part of cell cycle

1. Responsible for variation in cell cycle

(8 hrs to >100 days)
2. Cell’s irreversible decision to proliferate is
made here
- aided by cell division cycle proteins (CDC) which
helps cell to get beyond “restriction point”
- CDCs are protein kinases
 B. Gap Phase con’t.
3. Cell’s may enter a quiescent phase (Go) –
cells do not divide e.g. neurons & muscle cells

Factors that bring about quiescence

a) short supply of nutrients
b) “contact inhibition” – cell is in contact with
other cells
C. S (synthetic) phase – only period when DNA is
synthesized

Factors that induce DNA synthesis

2. Carcinogens and tumor viruses
3. Surgical removal of a tumor
4. Mitogens – proteins that bind to cell surface
receptors and induce cell division
 Types of Replication

1. Conservative replication – one daughter DNA

conserves the parental DNA while the other
daughter DNA gets the newly synthesized
DNA

2. Semiconservative replication – each daughter

DNA conserves one strand of parental DNA
and one strand of newly synthesized DNA
* type of replication in living organisms
Replication
fork
REPLICATION
formation of a new DNA strand using another
DNA as template
occurs at the S phase [cell cycle]
location: nucleus, mitochondrion & chloroplast in
eukaryotes
semi-conservative [Messelson & Stahl]
rate: 500 nts are added per second
few hrs to duplicate the human genome
error rate: 1 mispaired base in 109 nts
results to a 4n chromosome number
REPLICATION: Initiation
occurs at the oriC [E coli], ARS [fungi]
numerous origins in one chromosome
oriC: three 13-bp sequences - first to melt
five 9-bp sequences - binding sites for DnaA
proteins involved:
1. Topoisomerases - removes the supercoils
2. DnaA - ~ 30 units form a barrel → DNA wounds
3. DnaB - a Helicase; unwinds the DNA & breaks the
hydrogen bonds between the bases
4. DnaC - binds transiently to the DNA
5. SSB - prevents renaturation of the unwound DNA
RNA Primer - ~10 nts synthesized by Primase
REPLICATION: Elongation
5’

LAGGING STRAND

SSB Ligase
Okazaki fragment
3’ helicase
DNA Polymerase III

5’
DNA Polymerase I

primase

3’
Direction of replication fork
LEADING STRAND
REPLICATION: Elongation
DNA Polymerase III
polymerase function: 5’ → 3’ [α]
proofreading function: 3’ → 5’ exonuclease [ε]
Primase - synthesizes the RNA primer
DNA Polymerase I - replaces the primer with DNA
DNA Ligase - connects adjacent okazaki fragments
Eukaryotic DNA Polymerase
DNA Pol α - priming
DNA Pol β - DNA repair
DNA Pol γ - mitochondrial DNA replication
DNA Pol δ - main replicative enzyme
DNA Pol ε - unknown function
REPLICATION: termination
termination sites [Ter A - E] which are binding
sites for TUS [terminator utilization substances]

Shortening of the Telomere

Things to remember …...
replication starts at the oriC and ends at the Ter
DNA Polymerase III is the main replicative enzyme
direction of replication: 5’ → 3’
templates used: both strands
reqts for initiation: template, RNA primer, DNA Pol III
leading strand - replicated continuously
lagging strand - replicated discontinuously [okazaki]
linear chromosomes tend to shorten after replication
Tx: telomerase - not found in all cells
Fidelity: proofreading, excision repair, mutator proteins
Legend
Dna replication in action
Gene Transcription
(RNA Synthesis)
 Transcription

• Biosynthesis of RNA

• RNA copies of selected DNA sequences

(genes) are made
 Gene
• Classical definition
– A gene is a segment of DNA that
determines a single character or
phenotype
e.g., eye color, facial features,
height, etc.
 Gene

• Molecular definition
– A gene is a segment of DNA that
codes for:
• One enzyme
• One protein
• One polypeptide
• One RNA
 Types of Gene from Standpoint of
Function
A. Structural Genes
– Codes for polypeptides and RNA’s
B. Nonstructural Genes
Operator gene – one that serves as a
starting point for reading the genetic code
Regulator gene – one that synthesizes
the repressor (a substance which
switches off the activity of the structural
gene).
 Definition of Terms

• Cistron
– Smallest unit of DNA which must be intact
so that it can serve as transmitter of
genetic information
• Muton
– Smallest unit of DNA whose alteration can
give rise to a mutant form of organism
• Recon
– Smallest unit of DNA capable of
recombination, presumably a series of three
nucleotide bases (triplet).
• Recombination
– Formation of a new combination of genes
• Introns
– Intervening segments of DNA that do not
code for the amino acid sequence of the
polypeptide (function is probably regulatory)
• Exons
– Coding segments of the gene
• Chromosome
– A structure in the nucleus containing the
chromatin material made up of:
65% protein (histones)
35% DNA
5% RNA
– There are so many genes in a single
chromosome, e.g. E. coli = 3,000 to 5,000
genes
– Chromatin fibers resemble a string of beads
• Nucleosome
– Repeating beadlike structures composed of:
– Segment of DNA duplex (about 200
base pairs)
– Eight histone molecules (two each of
H2A, H2B, H3 and H4)
• Histones
– Found only in eukaryotes
– Function is to package and order DNA into
structural units called nucleosomes

– H1 = lysine-rich
– H2A and H2B = large amount of lysine
and arginine
– H3 and H4 = arginine-rich
TRANSCRIPTION

formation of RNA using a DNA as template

involves a short fragment of the genetic material only
only one of the 2 strands is used as template
direction of transcription: 5’ → 3’
location: nucleus, mitochondria, chloroplasts
occurs in most phases of the cell cycle
regulation: via Methylation of the CpG islands of genes
housekeeping genes are never methylated
rate: ~ 1,500 nts are transcribed in 50 seconds
TRANSCRIPTION
DNA-dependent RNA Polymerase [RNAP]
main enzyme involved in transcription
components: 1) core enzyme
- ααββ’
- polymerase function
2) sigma factor
- regulatory function

no proofreading capacity [less accurate than

DNA Polymerase III; prone to more errors]

Eukaryotic RNAPs:
RNAP I - transcribes rRNAs
RNAP II - transcribes mRNAs
RNAP III - transcribes tRNAs
A typical transcriptional unit

promoter Informative sequence terminator

DNA

Transcriptional start point

-100 -80 -10 +1 +N

GC CAAT TATA

TATA box - aka Pribnow box; recognition site of sigma factor

CAAT box - influences rate & frequency of transcription
GC box
octomers
A typical transcriptional unit

promoter Informative sequence terminator

DNA

GGCCGGCCGGCC------AAAAAAAAAAAAA

GC-rich region = allows hairpin loop formation

poly-A tract = allows dissociation of RNAP
A typical transcriptional unit

promoter Informative sequence terminator

DNA

Initiation codon Polyadenylation signal

Stop codon

Primary transcript

AAAAAAAAA
Mature mRNA
DNA before transcription

A C A T C G A C G C GC A C A
5’ 3’

3’ 5’
T G T A G C T G C G C G T G T
During transcription, The DNA should unwind so that one of its strand can be used as
template to synthesize a complementary RNA.
C A T C G A C G

A C A T C G A C G C GC A C A
5’ 3’
RNA
C A U C
5’ 3’

3’ 5’
T G T A G C T G C G C G T G T

G T A G C T G C
TRANSCRIPTION: steps

δ factor
RNAP

CLOSED promoter complex

OPEN promoter complex

Initiation

Elongation
TRANSCRIPTION
TRANSCRIPTION
Termination Signals
1) Rho-independent = GC-rich + poly-A tract
2) Rho-dependent = GC-rich + Rho protein

Modification of mRNAs
1) Capping = addition of 5’-methylguanosine cap;
occurs after the 5’ end has been synthesized
2) Polyadenylation = addition of the 3’-poly-A tail;
requires a clipping enzyme [5’-AAUAAA-3’]
and the Poly-A polymerase
3) Splicing of exons = removal of the introns and
splicing of the exons; follows the AG-TG rule
TRANSCRIPTION

Splice junctions: “AG-TG rule”

Exon 1 Exon 2
GT A AG

Intron
subsequent to capping & polyadenylation
prior to migration from the nucleus
req’ts: small nuclear ribonucleoprotein
particles [snRNPs] and snRNAs
[U1,U2, U4, U5 and U6]
COMPARISON
Replication Transcription
Location nucleus, mt, chpt nucleus, mt, chpt
Enzyme DNA Pol III RNAP
Direction 5’ → 3’ 5’ → 3’
Template both strands one strand
antisense
Primer needed not required
Target seq. entire genome a gene
Accuracy ↑↑↑ ↑
Units dNTPs: thymine NTPs: uracil
TRANSLATION

synthesis of a polypeptide based on an mRNA

template
location: cytoplasm [ribosomes]
requirements:

1) mRNA - with codons

2) tRNAs - with anti-codon
3) amino acids
main enzyme: peptidyl transferase
translational rate: ~ 15 amino acids/second
in E coli at 37o C
 GENETIC CODE
- maps DNA sequences to proteins in the living celL
- It is the sequence of bases (CODONS) in the
mRNA which determines the protein that will be
synthesized

CODON - 3 base words (43)

- 64 possible trinucleotide sequences are present
61 = code for amino acid
3 = stop codons
Starting codon = AUG
- marks the start of a protein sequence
End Codons = UAA, UAG, or UGA
 Properties of Genetic code
• Degenerate – amino acids are coded by several codon or some
codons may code for the same amino acid

e.g. UUA,UUG,CUU,CUC,CUA and CUG codes of leucine

• Specific – Each codon signals for a specific amino acid

• Non-overlapping and without punctuation/commaless
– the mRNA coding sequence is “read” by a ribosome starting
from the initiating codon (AUG) as a continuous sequence taken
three bases at a time until a stop codon is reached.
Reading frame – set of continuous triplet codons in a mRNA

• Universal – Examinations of the translation process in the species

that have been investigated have revealed that the coding signals for
amino acids are always the same. [though there are some
exceptions, especially in the mitochondrial and chloroplast DNA],
• Codons – Anticodon Interaction
The base pairing between the anticodon of the tRNA
and the mRNA codon is responsible for the actual
translation of the genetic information of structural
genes. Although codon-anticodon pairings are
antiparallel, both sequences are given in the 5’ -> 3’
direction.

ex. AUG binds to anticodon CAU

- the pairing of the codon (AUG) and anticodon

(CAU) ensures that the amio acid will be
incorporated into a growing polypeptide chain.

• Most cells possess about 50 tRNAs and some of the anticodon

contains uncommon nucleotides such as inosinate (I) which
typically occur at the third anticodon.
Codon-anticodon
mRNA
5’

3’

A=====U

tRNA
U=====A

G=====C

5’

3’
 WOBBLE HYPOTHESIS
- The first two bases are important in identifying their
cognate amino acids

- The third base [called the wobble base] is not as

important, pursuant to the Wobble theory of the
genetic code

- Wobble theory suggested that while the interaction

between the codon in the mRNA and the anticodon
in the tRNA needed to be exact in two of the three
nucleotide positions, this did not have to be so in the
third position

- The theory proposed that non-standard base-pairing

might occur between the nucleotide base in the 5'
position of the anticodon and the 3' position of the
codon.
5’ anticodon base 3‘ codon base

A U

C G

G C or U

U A or G

I A or C or U
TRANSLATION
Messenger RNA [mRNA]

Initiation codon Stop codon

SD AUG UGA PAS AAAA

codons

Shine Dalgarno Sequence - the ribosome binding site

AUG - initiation codon; contained within the Kozak
sequence in eukaryotes
Stop codons - UGA, UAG, UAA
PAS - Polyadenylation signal
NOTE: mRNA is derived from heterogeneous nuclear RNA
[hnRNA] in eukaryotes
TRANSLATION
Transfer RNA [tRNA]

cloverleaf appearance
adaptor molecule
~ 61 types of tRNAs

Pre-Initiation Phase: Charging of tRNA

TRANSLATION
Ribosomes
prokaryote eukaryote
small subunit 30S 40S
large subunit 50S 60S
complete unit 70S 80S

the small and large subunits combine after the initiation of

translation
small subunit = + mRNA binding site [16S RNA]
large subunit = + tRNA binding sites [P & A sites]
16S rRNA of the small subunit is highly
conserved
Initiation Step
1) Dissociation of 70S [IF1]

2) Binding of IF3 to 30S

3) Binding of IF1 & IF2-GTP

4) Formation of the Pre-

Initiation Complex

5) Binding of 50S to 30S &

loss of IF1 & IF3

6) Loss of IF2 and hydrolysis

of GTP →GDP + Pi
Elongation
1) Second aa-tRNA enters
the A site

2) Peptide bond formation

via Peptidyl transferase
[23S rRNA of the large
subunit]

3) mRNA moves to the left

Termination
occurs when a stop codon occupies the A site
UGA - “opal” codon
UAG - “amber” codon
UAA - “ochre” codon

no tRNA has the anticodon for the stop codons

a release factor recognizes these stop codons,
attaches to the A site and breaks up the complex

Release factor

AAAA
UGA
Post-translational Modification
Protein folding - proper folding of nascent polypeptide
made possible by molecular chaperones
HSP 70 - prevents premature folding
GroeL/GroeS - cage-like structures that prevents
aggregation of unfolded proteins

Intein Splicing - removal of inteins and the ligation of exteins

Inteins - intervening, non-informative aa sequences
Exteins - informative aa sequences

Signal peptide: ~ 20 aa, for proteins destined for

endomembrane system or for secretion; recognized by
the Signal Recognition Peptide [SRP; brings the ribosome to ER]

Proteolytic cleavage - polyproteins are cleaved to yield

several active proteins [ex. Proopiomelanocortin]
 ANTIBIOTICS BY SITE OF
ACTION
• A. Protein synthesis inhibitors
– 30S ribosomal subunit
• Aminoglycosides(streptomycin), tetracycline
– 50S ribosomal subunit
• Chloramphenicol – binds tp 50S and inhibits
peptidyl transferase
• Erythromycin and Clindamycin – binds to 50 S
and prevent translocation
 ANTIBIOTICS BY SITE OF
ACTION
• A. Protein synthesis inhibitors
– 60S ribosomal subunit
• Cycloheximide – binds to 60S,inhibits peptidyl
transferase
– A and P sites
• Puromycin – an amino acid analog, binds A
site,prevents eukaryotic and prokaryotic
translation
– eEF-2
• Diphtheria toxin – inhibits eukaryotic elongation
factor 2
 ANTIBIOTICS BY SITE OF
ACTION
• RNA synthesis inhibitors
– Prokaryotic RNA polymerase
• Actinomycin – binds to DNA
• Rifampin – binds to β-subunit of RNA
polymerase
– Eukaryotic RNA polymerase II
• Amanita phylloides- “angel of Death”
mushroom – inhibits RNA pol II
 ANTIBIOTICS BY SITE OF
ACTION
• DNA synthesis inhibitors
– DNA gyrase inhibitors
• Quinolones
– DNA topoisomerase inhibitors
• Nalidixic acid
• Novobiocin
 Other Medications: inhibits
deoxythymidylate synthesis
• 1. Folic acid analog
• inhibitor of dihydrofolate reductase
– Methotrexate
– Aminopterin
• 2. Inhibits Thymidylate Synthesis
– 5-Fluorouracil
LIST OF INHIBITORS
Coumarine inhibits DNA gyrase by competing with ATP
Quinolones inhibits DNA gyrase by binding its amino terminus
Rifampicin inihibits RNA polymerase by binding to its β chain
Chloramphenicol binds to 50S and prevents peptide bond formation
Erythromycin inhibits translocation of 50S subunit
Fusidic acid inhibits translocation by preventing dissociation of
EF-G-GDP from ribosome
Puromycin an aa-tRNA analog causing premature chain term’n
Tetracycline binds to 30S subunit and prevents aa-tRNA binding
to the A site
Cyclohexamide inhibits peptidyl transferase on eukaryotic 60S
Diphtheria toxin inhibits eukaryotic EF-2 by ADP ribosylation
Streptomycin binds to 30S subunit & causes mRNA misreading
Kanamycin binds to 70S unit and causes mRNA misreading
 mRNA

AUGUCUCCGUAA
Ribosome binding protein

Small ribosomal
subunit
Large ribosomal
subunit
 AUGUCUCCGUAA
Small ribosomal
Subunit binds to
mRNA
Large ribosomal
subunits binds to
small subunit
forming complete
ribosomes
AUGUCUCCGUAA

P – site on ribosomes

A – site on ribosomes
AUGUCUCCGUAA

UAC codons
Anti-codon

tRNA

Amino Acid (Met)

First, tRNA binds at
the P-site

AUGUCUCCGUAA

UAC

Anti-codon matches initiating codon on mRNA

AUGUCUCCGUAA
UAC

AGA

Next tRNA with

different anti-codon
and amino acid
AUGUCUCCGUAA
UAC
Second tRNA binds in the A
– site of the ribosome and
matches the next codon

AGA
AUGUCUCCGUAA
U A CA G A

Amino acid moved from

first tRNA and joined to
second tRNA forming a
di-peptide
 AUGUCUCCGUAA
U A CA G A

Ribosomes moves and the tRNA with di-

peptide moves to P-site

Empty tRNA is released

AUGUCUCCGUAA
AGA

GGC

Third tRNA with amino

acid arrives
AUGUCUCCGUAA Codon matches anti-codon
AGA

tRNA moves onto A-site

GGC

Third tRNA with amino

acid arrives
 AUGUCUCCGUAA
AGA GGC

Di peptide moved and Tri-peptide formed

 AUGUCUCCGUAA
AGA GGC
Ribosomes moves

tRNA moves to P-site

empty tRNA moves off ribosome

 AUGUCUCCGUAA
GGC
Ribosomes moves
Termination signal
no anti-codon on any tRNA
AGA

tRNA moves to P-site

empty tRNA moves off ribosome

AUGUCUCCGUAA
GGC

Complete polypeptide is released

AUGUCUCCGUAA
GGC

Ribosome units separate

Complete polypeptide is released

 What is the possible role of
modified histones?
A. methylation of histones is correlated with
activation and repression of gene
transcription
B. Methylation of histones leads to transcription
activation only
C. Phosphorylation of histones H3 and H4 is
associated with the condensation of
chromosomes during the replication cycle
D. Acetylation of histone H1 is associated with
chromosomal assembly during DNA
 What is the possible role of
modified histones?
A. methylation of histones is correlated with
activation and repression of gene
transcription
B. Methylation of histones leads to transcription
activation only
C. Phosphorylation of histones H3 and H4 is
associated with the condensation of
chromosomes during the replication cycle
D. Acetylation of histone H1 is associated with
chromosomal assembly during DNA
 Which is considered as
transcriptionally active chromatin
A. euchromatin
B. heterochromatin
C. constitutive chromatin
D. telomeres
 Which is considered as
transcriptionally active chromatin
A. euchromatin
B. heterochromatin
C. constitutive chromatin
D. telomeres
The genetic code is characterized
as:
A. non-degenerate
B. non-overlapping
C. species-specific
D. non-specific
The genetic code is characterized
as:
A. non-degenerate
B. non-overlapping
C. species-specific
D. non-specific
The precise initiation codon is
determined by:

A. shine-Dalgarno sequence
B. Kozak sequence
C. Consensus sequence
D. Repetitive sequence
The precise initiation codon is
determined by:

A. shine-Dalgarno sequence
B. Kozak sequence
C. Consensus sequence
D. Repetitive sequence
The first tRNA that enters the P site
during translation process.

A. Lys tRNA
B. Met tRNA
C. Ala tRNA
D. Glut RNA
The first tRNA that enters the P site
during translation process.

A. Lys tRNA
B. Met tRNA
C. Ala tRNA
D. Glut RNA
Factors that can cause DNA
denaturation:

A. Alkaline pH
B. High temperature
C. Acidic pH
D. A and B
E. B and C
Factors that can cause DNA
denaturation:

A. Alkaline pH
B. High temperature
C. Acidic pH
D. A and B
E. B and C
 Which enzyme is needed for
synthesis of the eukaryotic DNA
lagging strand
A. DNA polymerase alpha
B. DNA polymerase sigma
C. DNA polymerase III
D. DNA polymerase
 Which enzyme is needed for
synthesis of the eukaryotic DNA
lagging strand
A. DNA polymerase alpha
B. DNA polymerase sigma
C. DNA polymerase III
D. DNA polymerase
Which enzyme can remove the
supercoiling produced during
replication
A. Helicase
B. Topoisomerase
C. Primase
D. Single-strand binding protein
Which enzyme can remove the
supercoiling produced during
replication
A. Helicase
B. Topoisomerase
C. Primase
D. Single-strand binding protein
Matching Type:
__16. mRNA serves as template A. Transcription
__17. TATA Box plays a crucial role B. Translation
__18. May or may not require a rho facto C. Replication
__19. This is initiated with activation step D. A/B
__20. An essential process in cell divisio E. A/B/C
Matching Type:
_B_16. mRNA serves as template A.
Transcription
_A_17. TATA Box plays a crucial role B.
Translation
_A_18. May or may not require a rho facto C.
Replication
_B_19. This is initiated with activation step D.
A/B
_C_20. An essential process prior to division of cell E.
A/B/C
Any Questions ?