molecular biology presentation.pptx

Figure 11.1
Identical
base sequences
5’
5’
3’
3’ 5’
5’
3’
3’
Watson/Crick proposed mechanism of DNA replication

• In the late 1950s, three different mechanisms
were proposed for the replication of DNA
– Conservative model
• Both parental strands stay together after DNA replication
– Semiconservative model
• The double-stranded DNA contains one parental and one
daughter strand following replication
– Dispersive model
• Parental and daughter DNA are interspersed in both strands
following replication
Proposed Models of DNA Replication

Three models for DNA replication

• Matthew Meselson and Franklin Stahl
experiment in 1958
– Grow E. coli in the presence of 15N (a heavy isotope of
Nitrogen) for many generations
• Cells get heavy-labeled DNA
– Switch to medium containing only 14N (a light isotope of
Nitrogen)
– Collect sample of cells after various times
– Analyze the density of the DNA by centrifugation using
a CsCl gradient

CsCl Density Gradient Centrifugation
15N
14N
DNA

0
1
2
3
Generation
15N
Shift cells to 14N

Interpreting the Data
After one generation,
DNA is “half-heavy”
After ~ two generations, DNA
is of two types: “light” and
“half-heavy”
This is consistent with only
the semi-conservative model

Three main features of the DNA synthesis reaction:
1. DNA polymerase I catalyzes formation of phosphodiester bond
between 3’-OH of the deoxyribose (on the last nucleotide) and
the 5’-phosphate of the dNTP.
• Energy for this reaction is derived from the release of two of the
three phosphates of the dNTP.
2. DNA polymerase “finds” the correct complementary dNTP at each
step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate
3. Direction of synthesis is 5’ to 3’

DNA polymerase
Comparison of the properties of the DNA polymerase of E.Coli
Pol I Pol II Pol III
5’ → 3’ Polymerase Yes Yes Yes
3’ → 5’ Exonuclease Yes Yes Yes
5’ → 3’ Exonuclease Yes No No
Structure Polypeptide Poly peptide Multimeric complex
Function Repair, Primer excision Error Prone repair
polymerase
(SOS inducible)
Principle replication
polymerase
Subunits of E.Coli Pol III holoenzyme
Core Subunit
α 5’→ 3’ Polymerase activity, required for DNA synthesis
ε 3’→ 5’ exonuclease activity, required for proofreading
Q Function unknown.
Accessory
τ DNa dependent ATPase, required for initiation.
γ DNA dependent ATPase forming γ complex (with 4 peptides) facilitates β
subunit binding.
δ, δ1, χ, Ψ Forms γ complex required for loading & unloading β subunit
β ‘Sliding clamp’, forms preinitiation complex with DNA a process which
requires ATP dependent activity of the γ complex.

EUKARYOTIC DNA POLYMERASES
Mammalian Name α β γ δ ε
Yeast Name Pol 1 Pol 4 Pol M Pol 3 Pol 2
Yeast gene POL 1 POL 4 MIP 1 POL3 POL2
Location Nuclear Nuclear Mitochondria
l
Nuclear Nuclear
No. of subunit 4 1 2 2 >1
5’→3’Polymerase Yes Yes Yes Yes Yes
3’→ 5’
Exonuclease
No No Yes Yes Yes
Primase Yes No No No No
Associated
facotors
None None None PCNA None
Processivity Moderat
e
Low High High with
PCNA
High
Function Lagging
strand
priming
Repair
polymerase
Organelle
polymerase
Principle
replicative
polymerase
Unknown

Issues to Resolve for DNA Replication to
Occur
• Helix must be unwound
• Duplex over winding issues due to fork migration
• Primer synthesis
• One fork, two antiparallel strands…
• Primer removal
• Connecting the pieces…
• Proofreading and error correction

• Initiation of replication, major elements:
 Segments of single-stranded DNA are called template strands.
 Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.
 Initiator proteins and DNA helicase binds to the DNA at the
replication fork and untwist the DNA using energy derived from ATP
(adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)
 DNA primase next binds to helicase producing a complex called a
primosome (primase is required for synthesis),
 Primase synthesizes a short RNA primer of 10-12 nucleotides, to
which DNA polymerase III adds nucleotides.
 Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at
the RNA primer.
 The RNA primer is removed and replaced with DNA by polymerase I,
and the gap is sealed with DNA ligase.
 Single-stranded DNA-binding (SSB) proteins (>200) stabilize the
single-stranded template DNA during the process.

• DNA synthesis begins at a site termed the
origin of replication
• Each bacterial chromosome has only one
• Synthesis of DNA proceeds bidirectionally
around the bacterial chromosome
– eventually meeting at the opposite side of the
bacterial chromosome
• Where replication ends
BACTERIAL REPLICATION

Unwinding the Helix
• E. coli origin called oriC
– 245 bp region with repeating 9mer and 13mer repeating
sequences
• Each 9mer bound by several DnaA protein monomers
• DnaB and DnaC then bind
– Form a helicase that uses ATP to open helix
• Ssb protein binds to ss DNA
• Topoisomerase DNA gyrase eliminates positive
supercoiling caused by unwinding (uses ATP)

Initiation of Synthesis
• DNAPs cannot start new polynucleotide strand
– Must add on to something
• Primase (specialized RNAP) makes a short (5-15
nucleotide) RNA primer
– 3’end is used by DNAP III for new strand initiation
• RNA primer must later be removed and replaced by
DNA
– Done by DNAP I using 5’ exonuclease activity

RNA Primers for Initiation
• RNA primers for DNA synthesis are fairly universal in
their use
– E. coli, mammals, viruses/phage
– But not all viruses…

Initiation of Replication at oriC
• DNA replication is initiated by the binding of
DnaA proteins to the DnaA box
sequences
– causes the region to wrap
around the DnaA proteins and
separates the AT-rich region

Uses energy from ATP to
unwind the duplex DNA
SSB
SSB SSB
SSB

• DNA helicase separates the two DNA strands by
breaking the hydrogen bonds between them
• This generates positive supercoiling ahead of
each replication fork
– DNA gyrase travels ahead of the helicase and alleviates these supercoils
• Single-strand binding proteins bind to the
separated DNA strands to keep them apart
• Then short (10 to 12 nucleotides) RNA primers are
synthesized by DNA primase
– These short RNA strands start, or prime, DNA synthesis

Fig. 11.9a(TE Art)
Able to
covalently link
together
Unable to
covalently link
the 2 individual
nucleotides together
5’
5’
5’
5’
5’
5’
3’
3’
3’
3’
3’
DNA Polymerase Cannot Initiate new Strands

11-30
Figure 11.10
Innermost
phosphate

DNA Polymerase III- does the bulk of copying DNA in Replication

Figure 11.8 Schematic representation of DNA Polymerase III
Structure resembles a
human right hand
Template DNA thread
through the palm;
Thumb and fingers
wrapped around the DNA

Two dimensional view of a replication fork
Direction of synthesis
on leading strand
3’
5’
3’
5’
3’
5’

Figure 11.13 “Three Dimensional” view of Replication Fork
Direction of fork movement
Of lagging strand
of leading strand

Proofreading by the 3’  5’ exonuclease activity of DNA polymerases during DNA
replication.

DNA Duplexes Have Antiparallel
Strands…
• Replication forks move off from origins
– But to replicate both strands one DNAP or
replisome would seem to need to move back
toward the origin…
• Efforts to find a 3’ to 5’ DNAP for the “other”
strand failed
• One strand is made in a discontinuous fashion
(discontinuous or lagging strand)

Leading and
Lagging Strands
• Discontinuous synthesis
discovered by Okazaki
• Pulse-chase experiment
revealed that new DNA
was initially small pieces
but became very large
with time
• Right conclusion from
wrong results…

Lagging Strand Synthesis
• Lagging strand made as a series of 1000-2000
nucleotide pieces (bacteriophage), each
begging with an RNA primer (Okazaki
fragments)
• Primers removed by nick translation action of
DNAP I
• Fragments connected by DNA ligase
– Using NAD+ (E. coli) or ATP (eukaryotes and most
others)

Concurrent Synthesis of Leading
and Lagging Strands
• Kornberg model
– Process should be processive, not distributive
– Makes no sense for DNAP molecules to move away
from the fork and then have to return
– Has two DNAP III core enzymes connected to each
other
• One synthesizes each strand
– One continuously and one using a looping
discontinuous method that produces short Okazaki
fragments

3
Polymerase III
Leading strand
base pairs
5’
5’
3’
3’
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
Helicase
+
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2
Polymerase III
Lagging strand
Okazaki Fragments
1
RNA primer replaced by polymerase I
& gap is sealed by ligase

Fig. 3.8 Model of DNA Replication

Connecting Leading and Lagging Strand Synthesis Polymerase dimer synthesizes both
strands simultaneously using both a continuous and a looping discontinuous approach

Activities at the Replication Fork
• DNAP I and DNAP III, helicase (DnaBC, Rep), DNA
gyrase, SsB, primase, DNA ligase

Figure 11.4 Overview of bacterial DNA replication

Autoradiography: Radioactivity darkens film
Radioactive bacterial colonies on an agar petri dish

Directionality of the DNA strands at a replication fork
Leading strand
Lagging strand
Fork movement

Nicks are single strand breaks in double stranded DNA

Proofreading
• DNAPs have 3’exonuclease activity specific for single-stranded
DNA
– Unpaired nucleotides
• DNAPs “back up” to remove unpaired nucleotides and then
add correct base
– Improves fidelity of replication by 100X
– In E. coli activity in the e subunit of DNAP III

• DNA polymerases can only synthesize DNA only in the 5’ to 3’ direction and
cannot initiate DNA synthesis
• These two features pose a problem at the 3’ end of linear chromosomes
Figure 11.24 Problem at ends of eukaryotic linear Chromosomes

• If this problem is not solved
– The linear chromosome becomes progressively shorter with each round of
DNA replication
• The cell solves this problem by adding DNA
sequences to the ends of chromosome: telomeres
– Small repeated sequences (100-1000’s)
• Catalyzed by the enzyme telomerase
• Telomerase contains protein and RNA
– The RNA functions as the template
– complementary to the DNA sequence found in the telomeric repeat
• This allows the telomerase to bind to the 3’ overhang

Telomerase
• RNA-dependent DNA
polymerase
– Has its own RNA template
complementary to the telomeric
repeat sequence
– Adds on additional ss repeat
units
• Unusual hairpin structure
forms new primer to make ds
• Cleavage of hairpin yields
lengthened telomere

11-80
Figure 11.25
Step 1 = Binding
Step 3 = Translocation
The binding-
polymerization-
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Step 2 = Polymerization

DNA Repair mechanisms
• This section will review:
–The Role of DNA damage and its repair in the generation
of genetic diversity in bacteria
–The mechanistic links of repair with recombination
systems covered earlier
• You should be able to discuss the effect of environment on
damage and repair. For example:
–Chemical and Radiation effects
–Phagocytic damage in relation to pathogen survival in the
host
–Errors in replication and their repair.

How can DNA become damaged?
• Mismatched bases
– Polymerase error rate about 1 in 104 (see later lectures)
– Deamination of C to U leading to mismatch
• Missing bases. Hydrolysis of purine-deoxyribose bond
leading to AP-site.
• Structural damage. Dimer formation.
• Broken phosphodiester bonds. Chemicals/radiation
• REPAIR MECHANISMS NECESSARY FOR SURVIVAL.

Types of DNA Damage Summarised
G A C
T
ds DNA Break Mismatch
Thymidine dimer
AP site
Covalent X-linking
ss Break
C-U deamination

General repair mechanisms needed
• EITHER Reverse damage (e.g. PHOTOREACTIVATION)
• OR excise DNA and patch repair the region
Photoreactivation: Discovered in Actinomycetes in 1949
UV - DNA Damage - Cell Death
UV - Bright visible light - survival !
3 Steps:
•Photolyase (encoded by phrA and phrB genes in E. coli) recognises
distortion at dimer.
•Light activates photolyase
•Dimer cleaved

General repair mechanisms needed: Excision
repair.
• Discovered first as a general mechanism in 1964
• T- Phage HOST CELL REACTIVATION
T-Phage suspension
DNA Damage
Repair
-
WT
Repair
Plaques !
UV

Excision repair........
5’
3’
3’
5’
5’
3’
3’
5’
OR
5’
3’
3’
5’
UvrABC
5’
3’
3’
5’
Pol1
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’

Other repair routes.
•Excision repair involves up to 20 nucleotides
•uvrA,BC (D) mutants very senstive to UV light
•Mismatch repair
•A from of excision repair. Dam methylase involved
•see later re: methylation
•N-glycolylase excision repair
•Uracil either misincorporated OR C deaminated to U
•Uracil N-glycosylase action TO give AP site
•AP endonuclease cut
•Patch repairs above

How does UV light cause mutations? Discovery
of error-prone repair.
• RecA required for high level of UV mutagenesis
•UV dose of 2 µJ/mm2 leads to 120 dimers
•Long patch Error-prone repair
•Post dimer initiation
•Trans dimer synthesis
The SOS Hypothesis
Radman 1974 originally proposed an inducible repair
system
Requires RecA and a regulator system

SOS System in E.coli
• Repair normally at low level
• lexA gene identified as a regulator
• Recombine normally
• But NO increased UV mutagenesis (ie 30 dimers produces
no extra mutants). Higher doses required
• LOW DOSE - Error-free repair
• HIGH DOSE - Error repair INDUCED
• LexA is an autoregulated repressor
• Represses level of activity of many genes
• Collectively called DNA Inducible (din) genes
• Includes uvrA,B,C,D and sfi etc...
• RecA protease activity; Cleaves LexA
• Also CI repressor inducing lysis

SOS System in E.coli
PO lexA PO recA
PO din
PO din
PO din
PO din
PO din
PO din
PO din
PO din
PO din
PO din
Low level expression
PO lexA PO recA
HIGH level expression

Post Replication / Recombination Repair
•recA mutants VERY UV sensitive
•uvrA similarly
•uvrA recA mutants VERY VERY UV sensitive
•recA mutants recover slowly in the dark
•uvrA mutants do not
UV dose Dimers/
Genotype Phenotype µJ/mm2 genome
WT WT 5.0 3200
uvrA No excision repair 0.8 50
recA No recombination 0.3 20
recA, uvrA Neither 0.02 1

Post Replication / Recombination Repair
Stephen C. Kowalczykowski (2000) Initiation of genetic
recombination and recombination-dependent
replication. TIBS 25 – April 2000
• Double-stranded-break repair – see transposition of
Tn10 later
• Recombination-dependent replication
• Replication-dependent recombination
SEE ALSO Key Reference in NATURE; STRUCTURE OF RecBCD
complex
• Nature 11th Nov 2004 vol 432, 187-193

RNA polymerase in bacteria
In bacteria, the same enzyme catalyzes the synthesis of three types of RNA: mRNA,
rRNA and tRNA.
RNAP is a relatively large molecule. The core enzyme has 5 subunits (~400 kDa):
• α2: the two α subunits assemble the enzyme and recognize regulatory factors.
• β: this has the polymerase activity (catalyzes the synthesis of RNA).
• β’: binds to DNA (nonspecifically).
• ω: function not known clearly.
RNA polymerase in eukaryotes
Eukaryotes have several types of RNAP:
• RNA polymerase I synthesizes a pre-rRNA 45S, which matures into 28 S, 18S and
5.8S rRNAs which will form the major RNA sections of the ribosome.
• RNA polymerase II synthesizes precursors of mRNAs and most snRNA. This is the
most studied type, and due to the high level of control required over transcription
a range of transcription factors are required for its binding to promoters. For detail
of RNA polymerase function please see RNA polymerase II.
• RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the
nucleus and cytosol.
Other RNA polymerase types in mitochondria and chloroplasts.

THE lac OPERON
© 2007 Paul Billiet ODWS

The control of gene expression
• Each cell in the human contains all the genetic
material for the growth and development of a
human
• Some of these genes will be need to be expressed all
the time
• These are the genes that are involved in of vital
biochemical processes such as respiration
• Other genes are not expressed all the time
• They are switched on an off at need

Operons
• An operon is a group of
genes that are
transcribed at the
same time.
• They usually control an
important biochemical
process.
• They are only found in
prokaryotes.
© NobelPrize.org
Jacob, Monod & Lwoff

The lac Operon
 The lac operon consists of three genes each
involved in processing the sugar lactose
 One of them is the gene for the enzyme β-
galactosidase
 This enzyme hydrolyses lactose into glucose
and galactose

Adapting to the environment
• E. coli can use either glucose, which is a
monosaccharide, or lactose, which is a
disaccharide
• However, lactose needs to be hydrolysed
(digested) first
• So the bacterium prefers to use glucose when
it can

Four situations are possible
1. When glucose is present and lactose is absent the E. coli
does not produce β-galactosidase.
2. When glucose is present and lactose is present the E. coli
3. When glucose is absent and lactose is absent the E. coli
4. When glucose is absent and lactose is present the E. coli
does produce β-galactosidase

1. When lactose is absent
• A repressor protein is continuously synthesised. It sits on a
sequence of DNA just in front of the lac operon, the Operator
site
• The repressor protein blocks the Promoter site where the
RNA polymerase settles before it starts transcribing
Regulator
gene
lac operon
Operator
site
z y a
DNA
I
O
Repressor
protein
RNA
polymerase
Blocked

2. When lactose is present
• A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
• This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
z y a
DNA
I O

2. When lactose is present
• A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
• This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
Promotor site
z y a
DNA
I O

3. When both glucose and lactose are
present
• This explains how the lac operon is
transcribed only when lactose is present.
• BUT….. this does not explain why the operon
is not transcribed when both glucose and
lactose are present.

• When glucose and lactose are present RNA
polymerase can sit on the promoter site but it is
unstable and it keeps falling off
Promotor site
z y a
DNA
I O
Repressor protein
removed
RNA polymerase

4. When glucose is absent and lactose is
present
• Another protein is needed, an activator protein. This
stabilises RNA polymerase.
• The activator protein only works when glucose is absent
• In this way E. coli only makes enzymes to metabolise other
sugars in the absence of glucose
Promotor site
z y a
DNA
I O
Transcription
Activator
protein steadies
the RNA
polymerase

Summary
Carbohydrates Activator
protein
Repressor
protein
RNA
polymerase
lac Operon
+ GLUCOSE
+ LACTOSE
Not bound
to DNA
Lifted off
operator site
Keeps falling
off promoter
site
No
transcription
+ GLUCOSE
- LACTOSE
Not bound
to DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
- LACTOSE
Bound to
DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
+ LACTOSE
Bound to
DNA
Lifted off
operator site
Sits on the
promoter site
Transcription

Negative Regulation
X
Positive Regulation
X

NEGATIVE REGULATION
X
INDUCIBLE TRANSCRIPTION
X
REPRESSIBLE TRANSCRIPTION
X

X
NEGATIVE REGULATION
INDUCIBLE TRANSCRIPTION
X
REPRESSOR
INDUCER
INACTIVE
REPRESSOR
REPRESSOR BINDING SITE
OPERATOR
The lac Operon

The lac Operon
P O lacZ lacY lacA
P O lacZ lacY lacA
mRNA 5’ 3’
RIBSOSOME BINDING SITE
Q: How many proteins are made?

The lac Operon
P O lacZ lacY lacA
P O lacZ lacY lacA
mRNA 5’ 3’
Proteins
b-galactosidase Permease Transacetylase

The lac Operon
-b-galactosidase and permease encode proteins that metabolize lactose to give
glucose.
-In the absence of lactose in the medium, the genes are turned off. That is, no
mRNA is transcribed and no proteins are made.
-In the presence of lactose in the medium, the genes are turned on. That is, mRNA
is transcribed and the proteins are made.
X
X
REPRESSOR
INDUCER
INACTIVE
REPRESSOR
OPERATOR
Q: Which molecule do you think is the inducer?

The lac Operon
P O lacZ lacY lacA
lac repressor
X NO mRNA
lac repressor
-Protein that is encoded by the lacI gene.
-The lacI gene has its own promoter.
-The lac repressor binds to the operator and inhibits transcription of the lac operon.

The lac Operon
In the presence of lactose in the medium, lactose binds to the repressor.
The lactose-repressor complex is unable to bind to the operator.
P O lacZ lacY lacA
X NO mRNA
lactose
P O lacZ lacY lacA
X NO mRNA

The lac Operon
The RNA polymerase can now bind to the promoter and initiate
transcription of the genes. The proteins made metabolize the lactose.
P O lacZ lacY lacA
mRNA

NEGATIVE REGULATION
X
Aporepressor
Co-repressor
Active repressor

NEGATIVE REGULATION
X
Aporepressor
Co-repressor
Active repressor
Operator
THE trp OPERON

X
Aporepressor
Co-repressor
Active repressor
Operator
THE trp OPERON
-In the presence of tryptophan in the cell, the genes are turned off. That is,
mRNA is not transcribed and the proteins are not made.
-In the absence of tryptophan in the medium, the genes are turned on. That
is, mRNA is transcribed and proteins are made.
Q: Which molecule is the co-repressor?

Positive Regulation
X
Transcriptional Activator
Enhancer

Transcriptional Activators
- Are proteins that activate transcription (No Dah!).
- Bind to enhancers (in eukaryotes).
- Their activity is modulated by environmental conditions.
Enhancers (in eukaryotes)
- Are specific DNA sequences that bind to transcriptional activators.
- Can be found upstream or downstream of the promoter.
- Do not need to be next to promoter.

GENRAL NOTES
1) Positive and negative regulation is used for all kinds of
genes, polycistronic and monocistronic. It is used in all
organisms.
2) Most genes have both positive and negative regulation
(for example, lac operon).
3) When transcription is repressed, there is a very low level
of mRNA made. That is called basal transcription.

E. coli changes gene expression to utilize
different nutrient sources

Lac Operon (Negative Regulation)
Catabolic Enzyme
Repressor
Inducer
Operon

The lac repressor dissociates from the operator
sequence upon IPTG (inducer) binding

Cis versus Trans
Dominant versus Recessive

Operator Mutations Reduce Repressor
Binding

Fig. 7.12
The lac repressor tetramer binds two operators

Figs. 7.16 & 7.13
The lac operon is also regulated by glucose levels
Low Glucose => High cAMP
=> activation of lac operon thru CAP binding site
Glucose
is
Sweeter

cAMP is Needed for
Activation
Catabolite Repression

Figs. 7.17 & 7.19
CAP-cAMP binding creates DNA bending and
activates lac operon transcription

Trp Operon
(Anabolic Enzyme)
Apo
Co-repressor

Figs. 7.28 &7.31
The trp operon attenuator

Termination
No termination
Fig. 7.30
Alternative
structures of the
trp operon
attenuator RNA

Reading: MVA pp. 760-767 and 1011-1018
Problem Ch 26: 9
Alberts: pp. 395-399

The chromosomal order of genes in the trp operon of E. coli and the sequence of
reactions catalyzed by the enzyme products of the trp structural genes. The products
of genes trpD and trpE form a complex that catalyzes specific steps, as do the
products of genes trpB and trpA. Tryptophan synthetase is a tetrameric enzyme
formed by the products of trpB and trpA. It catalyzes a two-step process leading to
the formation of tryptophan. (PRPP, phosphoribosyl pyrophosphate; CDRP, 1-(o-
carboxyphenylamino)-1-deoxyribulose 5-phosphate.) (After S. Tanemura and R. H.
Bauerle, Genetics 95, 1980, 545.)

Originally, regulation of the trp operon was thought to occur solely through the
repressor-operator system until deletion mutants located downstream of trpO were
identified. These mutants displayed increased expression of the operon by six-fold
which indicated the presence of an additional transcriptional control element.
Why is repression not the only mode of regulation?

vidence:
1. Trp-tRNA synthetase mutants had regulatory anomalies.
2. Addition of trp to trp-starved cells not only shut down
initiation of transcription but also inhibited transcription
already in progress on the initial segment of the operon.
3. Mutants lacking a functional repressor could still respond
to trp starvation by increasing transcription of trp mRNA.
4 . Deletion mutants in which both of the deletion termini were
within the transcribed region of the operon had an
unexpected six-fold increase in expression of the remaining
genes in the operon. Obviously, repressor binding was
unaffected.

5. Within the population of mRNAs produced in vivo from the
5' end of the trp operon, RNAs corresponding to the first
140 bp (the leader sequence) of the operon were several times
more abundant than those from more distal regions, therefore
a transcription termination site was located before the structural
genes.
6. Starving bacteria of trp reduced termination at this site
(the trp attenuator).
7. Mutations altering trp-tRNA synthetase, tRNAtrp or a
tRNA trp modifying enzyme were found to decrease transcription
termination at the trp attenuator. What does this suggest
about the mode of attenuation?

8. Ribosome binding experiments with the 140 base transcript
demonstrated that ribosomes protect a 20 base segment from
nuclease attack. A potential AUG start codon is located in the
center of this region.
9. A 14 residue peptide (the leader peptide) could be
synthesized from this start codon and contained tandem trp
residues near its C-terminus.
10. The trp leader ribosome binding site was shown to be an
efficient site for the initiation of translation by fusing the leader
to a structural gene and demonstrating synthesis of the fused
polypeptide.

11. Two classes of termination defective leader mutants have
been isolated. One type terminates at less than normal
frequency and has bp changes in the 3:4 bp region. In vivo,
these mutants have a 2-4 fold increase in operon expression.
12. The second class of mutants have increased termination of
the attenuator. These prevent the relief from termination that
is associated with trp starvation. One of these mutants has an
altered start codon for the leader peptide. Another has a
G to A conversion at position 75, which would prevent
2:3 pairing and cause formation of a 3:4 termination structure.

Genetic analysis indicated that the new control element was
located in trpL, a 162 nt region 30-60 nt upstream from trpE.
When trp is scarce, the entire 6720 nt polycistronic trp, including
trpL, is synthesized. As the trp concentration increases, the rate
of trp transcription decreases as a result of the trp r
epressor-corepressor's greater abundance.
With increasing [trp], the mRNA synthesized consists more and
more of a 140 nt segment corresponding to trpL sequences only.
The availability of trp results in the premature termination of
transcription of the operon.

How a Terminator Loop is Formed

Lambda can Lyse bacterial Cells

Anti-terminator - A Friend in Disguise
N

How to Start the Feedback Loop
CI
CI/CIII

Feedback Loop is Maintained
Lamda lives Happily Thereafter…
CI

Lysogeny versus Lysis
CI wins -
Cro wins -
Lysogeny
Lytic

What If Stock Market Turns South ?
SOS
RecA+
Lambda
Protease

a-Helix Recognizes DNA Major Groove

DNase Footprinting Reveals Site of Binding

Amino-acid Side-chains Contact
Functional Groups of Nucleotides

Major Groove -
A Rich Source of
Specificity

Stages of Transcription - Initiation

Stages of Transcription – Elongation
and Termination

Bacterial Promoters
• A promoter is where RNApoly binds
• Template strand versus Coding strand
• Upstream – in the 5’ direction on the coding
strand
• Consensus sequences

Consensus Sequences
AGGTTAGCGGATCGATCGATGTGACAAGGATATATGCAGTC
CGATTAGCCGATACGCAATTTTGACAAGGATATATATGCAG
CGCTTAGCGGATGGCAGAAAGAGACAAGGATATATGAATTC
CGATTAGCGGATTCCTTCCCACGACAAGGATATATCGACGA
CAGTTAGCGGATTTTACGATCAGACAAGGATATATTAACGA
---TTAGCGGAT----------GACAAGGATATAT------

Initiation
• Stayes put for a bit
• Nucleoside triphospates (NTPs) are added one by one until 9 bp
have been added, and then elongation can occur (i.e. RNApoly
can move)

Elongation
• Unwinding of DNA
• Adding NTPs
• About 12 bp of RNA are bonded to DNA as it goes
• DNA is rewound behind
• RNApoly moves along the template strand from 3’ to 5’, thus RNA elongates from the 5’ to 3’
end

Termination
• The terminator is in
the transcript, not
the DNA
• Forms a hairpin
• Self-
complementary
• The hairpin
structure is the
signal for
termination

Eukaryotic transcription
• Three RNA polymerases
• One for each major type of RNA
– RNApoly I - makes pre-rRNA
– RNApoly II - makes pre-mRNA
– RNApolyIII - makes pre-tRNA
• Each polymerase has a different promoter
structure

RNApoly II promoter
• Initiator sequence surrounding the start point
• TATA box at about –25 bp
• TATA+Initiator = core promoter
• A transcription factor binds to the TATA box
before RNAPoly II

Transcription factors
 A basal transcription factor is
always required to allow
RNApoly to bind to DNA
 For RNApoly II, TFIID binds to
the TATA box. This is the basal
transcription factor.
 More TFs bind to TFIID through
protein-protein interactions to
form the pre-initiation
complex.
 Then RNApoly binds
 Many TFs may be involved

mRNA modifications
• The primary transcript is modified
• 5’ caps
• poly(A) tails
• splicing

mRNA Modifications: 5’methylated
cap
• A “backwards” 5’ cap of methylated guanine
• Added during elongation
• Functions
– Flag for nuclear export
– Protect against degradation
– Binding site for the ribosome
• Catalyzed by a "capping enzyme"
that only associates with RNA
poly II
• Why might this be important?

mRNA modifications: polyadenylation
• Transcripts are generally too long
• poly(A) polymerase (PAP) finds a poly(A) signal
(AAUAAA) that marks the end of the important
stuff.
• PAP cleaves the transcript.
• PAP then adds a polyA tail
to the newly cleaved mRNA
• The poly-A tail:
– helps protect the transcript
from degradation
– is necessary for full
initiation of translation

mRNA degradation and poly(A) tails
G
XRN
• De-adenylation promotes rapid
degradation
DCP

Exons usually represent protein
domains

Splicing
• Introns need to be removed from the
primary transcript

Spliceosome
• Splicing introns from mRNA occurs at short,
conserved sequences called splice sites which
specify the beginning and ends of introns.
• GU on the 5' end and AG on the 3' end are
100% conserved.
• Catalyzed by a structure called a spliceosome,
composed of protein and RNA (snRNA – small
nuclear RNA)

Regulation of transcription
• Gene “expression” and subsequent protein
production is controlled primarily at the level
of transcription.
• That is, if you transcribe the gene, you will
make the protein.
• MODY is a defect in insulin gene regulation,
not in transcription per se.

Example in bacteria
Goal: Express the enzymes
necessary to use lactose as an
energy source only if lactose is
present in the environment

Operon model
• Operon: Several genes with related functions that
are regulated together, because one piece of
mRNA codes for several related proteins.
• polycistronic mRNA,, mRNA coding for more than
one polypeptide, is found only in prokaryotes

Operator
• The operator is a sequence located between
the promotor and the first structural gene
• Proteins may bind to the operator and
promote or inhibit transcription.

Operon model
• An operon consists of:
– Structural genes - code for the
enzymes/proteins of interest.
– Regulatory genes - control the expression of
structural genes by expressing regulatory
proteins

The lac operon
• Repressors – bind to the operator and inhibit
• Negative control of transciption, because binding of a
protein to the operator turns transcription off

The lac operon
• Substrate induction, or operon de-
repression

Example in bacteria
• The lac operon is involved with utilizing
lactose as an energy source (a catabolic
pathway).
• Anabolic pathways are also controlled
• Example: stop manufacturing the amino acid
tryptophan if you have enough of it, either
from synthesis or from the environment.

The trp operon
• In the presence of tryptophan, the repressor
for the trp operon binds to the operator.
• Example of end product repression

Review…
• In substrate induction, substrate (effector)
binding to the repressor renders it unable to bind
to the operator, and allows transcription to
initiate.
• In end-product repression, product (effector)
binding to the repressor makes it bind. This
prevents initiation of transcription.
• These are both negative forms of regulation

• Glucose pathways are
constitutively expressed
• If glucose is present, turn
down other pathways (like
lactose catabolism)
• If absent, only turn up
those pathways for which
there are substrates (like
lactose)
• Example: CRP in lac that
promotes polymerase
binding!
Glucose  = cAMP 
Positive control of the lac operon

Test yourself…
• What would happen in the lac operon if…
– There IS glucose, and there is NOT lactose?
– There is NOT glucose, and there IS lactose?
– There IS glucose, and there IS lactose?

A few more terms…
• Regions in/around the promoter to which
regulatory proteins may bind are called cis-
acting elements.
• Regulatory genes are often called trans-acting
elements, because they can exist far away on
the DNA
– Their products, regulatory proteins, diffuse and
bind to the cis-elements.
– The gene for a repressor is one example.

Regulatory elements in eukaryotes
• The basic principles that control transcription in
bacteria also apply to eukaryotic organisms:
– controlled by trans-acting proteins (transcription
factors) binding to cis-acting DNA sequences
• However,
– eukaryotic cis-acting elements are often much
further from the promoter they regulate,
– Any given transcription factor may be involved as
one of many TFs in the transcription of many
different genes.

Complex control of eukaryotic genes
Trans-acting element
TFA
TFA
TFA
Trans-acting element
TFB
TFB

Proximal control elements
• The core promoter can typically turn on
transcription only at a low rate. Add to these…
• Proximal control elements
– sequences <100 bases upstream of the core
promoter to which transcription factors bind and
improve the efficiency of the core promotor.
• Transcription factors that bind outside the
core promotor are called regulatory
transcription factors.

• > 100 bp upstream
or downstream
• Enhancers
– TFs that bind here
are activators
• Silencers
– TFs that bind here
are repressors
Distal control elements

Steroid hormones
• Steroid hormones pass
through the cell
membrane
• Bind to and activate
(or un-inhibit) a steroid
receptor
• The receptor moves
to the nucleus
• The receptor acts as
a transcription factor

The genetic code: how do nucleotides specify 20 amino acids?
1. 4 different nucleotides (A, G, C, U)
2. Possible codes:
• 1 letter code  4 AAs <20
• 2 letter code  4 x 4 = 16 AAs <20
• 3 letter code  4 x 4 x 4 = 64 AAs >>20
3. Three letter code with 64 possibilities for 20 amino acids suggests that the
genetic code is degenerate (i.e., more than one codon specifies the same
amino acid).

The genetic code is a triplet code
A set of 3 consecutive nucleotides make a codon in mRNA code, which corresponds to
one amino acid in a polypeptide chain.
1. 1960s: Francis Crick et al.
2. Studied frameshift mutations in bacteriophage T4 (& E. coli), induced by the
mutagen proflavin.
3. Proflavin caused the insertion/deletion (indels) of a base pair in the DNA.
4. Two ways to identify mutant T4:
1. Growth with E. coli B:
• r+(wild type)  turbid plaques
• rII (mutant)  clear plaques
2. Growth with E. coli K12 ():
• r+ (wild type)  growth
• rII (mutant)  no growth

1. Discovered that frameshift mutations (insertion or deletion) resulted in a
different sequence of amino acids.
2. Also discovered that r+ mutants treated with proflavin could be restored to
the wild type (revertants).
Fig. 6.5

3. Combination of three r+ mutants routinely yielded revertants, unlike other
multiple combinations.
Fig. 6.6 - Three nearby insertions (+) restore the reading frame, giving normal or
near-normal function.

How was the genetic code deciphered?
1. Cell-free, protein synthesizing machinery isolated from E. coli. (ribosomes, tRNAs,
protein factors, radio-labeled amino acids).
Synthetic mRNA containing only one type of base:
UUU = Phe, CCC = Pro, AAA = Lys, GGG = ? (unstable)
2. Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA, AAC, AAA) composed of
two different bases:
Pro, Lys (already defined) + Asp, Glu, His, & Thr
Proportion (%AC) varied to determine exactly which codon specified which
amino acid.
3. Synthetic polynucleotides of known composition:
UCU CUC UCU CUC  Ser Leu Ser Leu
1968: Robert Holley (Cornell), H. G. Khorana (Wisconsin-Madison),
and Marshall Nirenberg (NIH).

How was the genetic code deciphered (cont.):
4. Ribosome binding assays of Nirenberg and Leder (1964) (ribosomes, tRNAs
charged w/AAs, RNA trinucleotides).
 Protein synthesis does not occur.
 Only one type of charged tRNA will bind to the tri-nucleotide.
mRNA UUU codon
tRNA AAA (with Phe) anti-codon
mRNA UCU codon
tRNA AGU (with Ser) anti-codon
mRNA CUC codon
tRNA GAG (with Leu) anti-codon
 Identified 50 codons using this method.
5. Combination of many different methods eventually identified 61 codons, the
other 3 do not specify amino acids (stop-codons).

Characteristics of the genetic code (written as in mRNA, 5’ to 3’):
1. Code is triplet. Each 3 codon in mRNA specifies 1 amino acid.
2. Code is comma free. mRNA is read continuously, 3 bases at a time without
skipping bases (not always true, translational frameshifting is known to occur).
3. Code is non-overlapping. Each nucleotide is part of only one codon and is read
only once.
4. Code is almost universal. Most codons have the same meaning in different
organisms (e.g., not true for mitochondria of mammals).
5. Code is degenerate. 18 of 20 amino acids are coded by more than one codon.
Met and Trp are the only exceptions. Many amino acids are four-fold degenerate
at the third position.
6. Code has start and stop signals. ATG codes for Met and is the usual start signal.
TAA, TAG, and TGA are stop codons and specify the the end of translation of a
polypeptide.
7. Wobble occurs in the tRNA anti-codon. 3rd base is less constrained and pairs less
specifically.

Wobble hypothesis:
 Proposed by Francis Crick in 1966.
 Occurs at 3’ end of codon/5’ end of anti-codon.
 Result of arrangement of H-bonds of base pairs at the 3rd pos.
 Degeneracy of the code is such that wobble always results in translation of the
same amino acid.
 Complete set of codons can be read by fewer than 61 tRNAs.
5’ anti-codon 3’ codon
G pairs with U or C
C pairs with G
A pairs with U
U pairs with A or G
I (Inosine) pairs with A, U, or C
I = post-transcription modified purine
Fig. 6.8

TTT TCT TAT TGT
TTC TCC TAC TGC
TTA TCA TAA TGA
TTG TCG TAG TGG
CTT CCT CAT CGT
CTC CCC CAC CGC
CTA CCA CAA CGA
CTG CCG CAG CGG
ATT ACT AAT AGT
ATC ACC AAC AGC
ATA ACA AAA AGA
ATG ACG AAG AGG
GTT GCT GAT GGT
GTC GCC GAC GGC
GTA GCA GAA GGA
GTG GCG GAG GGG

PHE
PHE
SER
SER
SER
SER
TYR
TYR
CYS
CYS
LEU
LEU
LEU
LEU
LEU
LEU
STOP
STOP
STOP
TRP
PRO
PRO
PRO
PRO
HIS
HIS
ARG
ARG
ARG
ARG
GLN
GLN
ILE
ILE
ILE
THR
THR
THR
THR
ASN
ASN
SER
SER
LYS
LYS
ARG
ARG
MET
VAL
VAL
VAL
VAL
ALA
ALA
ALA
ALA
ASP
ASP
GLY
GLY
GLY
GLY
GLU
GLU

PHE
PHE
SER
SER
SER
SER
TYR
TYR
CYS
CYS
LEU
LEU
LEU
LEU
LEU
LEU
STOP
STOP
STOP
TRP
PRO
PRO
PRO
PRO
HIS
HIS
ARG
ARG
ARG
ARG
GLN
GLN
ILE
ILE
ILE
THR
THR
THR
THR
ASN
ASN
SER
SER
LYS
LYS
ARG
ARG
MET
VAL
VAL
VAL
VAL
ALA
ALA
ALA
ALA
ASP
ASP
GLY
GLY
GLY
GLY
GLU
GLU
NEUTRAL-NONPOLAR
NEUTRAL-POLAR
BASIC
ACIDIC

Evolution of the genetic code:
 Each codon possesses an inherent set of possible 1-step amino acid changes
precluding all others.
 As a result, some codons are inherently conservative by nature, whereas others
are more radical.
 Phe, Leu, Ile, Met, Val (16 codons with T at 2nd pos.) possess 104 possible
evolutionary pathways.
 Only 12 (11.5%) result in moderately or radically disimilar amino acid
changes
 Most changes are nearly neutral because they results in substitution of
similar amino acids.
 DNA sequences with different codons compositions have different properties,
and may evolve on different evolutionary trajectories with different rates of
substitution.

Evolution of the genetic code (cont.):
 On average, similar codons specify similar amino acids, such that single base
changes result in small chemical changes to polypeptides.
 For example, single base changes in the existing code have a smaller average
effect on polarity of amino acids (hydropathy/hydrophily) than all but 0.02% of
randomly generated genetic codes with the same level of degeneracy
(Haig and Hurst 1991, J. Mol. Evol. 33:412-417).
 The code has evolved to minimize the severe deleterious effects of substituting
hydrophilic for hydrophobic amino acids and vice versa (this also is true for other
properties).
 This is a good thing!!!

Translation-protein synthesis (Overview):
1. Protein synthesis occurs on ribosomes.
2. mRNA is translated 5’ to 3’.
3. Protein is synthesized N-terminus to C-terminus.
4. Amino acids bound to tRNAs are transported to the ribosome. Facilitated by:
 Specific binding of amino acids to their tRNAs.
 Complementary base-pairing between the mRNA codon and the tRNA anti-
codon.
 mRNA recognizes the tRNA anti-codon (not the amino acid).

Translation - 4 main steps
1. Charging of tRNA
2. Initiation
3. Elongation (3 steps)
1. Binding of the aminoacyl tRNA to the ribosome.
1. Formation of the peptide bond.
1. Translocation of the ribosome to the next codon.
4. Termination

Step 1-Charging of tRNA (aminoacylation)
1. Amino acids are attached to tRNAs by aminoacyl-tRNA synthetase and Produces
a charged tRNA (aminoacyl-tRNA).
2. Uses energy derived from ATP hydrolysis.(ATP=AMP+PPi)
3. 20 different aminoacyl-tRNA synthetases (one for each AA).
4. tRNAs possess enzyme-specific recognition sites for aminoacyl-tRNA synthetase
5. Sequence of events:
1. ATP and amino acid bind to aminoacyl-tRNA synthetase, to form aminoacyl-
AMP + PPi.
2. tRNA binds to aminoacyl-AMP.
3. Amino acid transfers to tRNA, displacing AMP.
4. Amino acid always is attached to adenine on 3’ end of tRNA by its carboxyl
group forming aminoacyl-tRNA.

Step 2-Initiation-requirements:
1. mRNA
2. Ribosome
3. Initiator tRNA (fMet tRNA in prokaryotes)
4. 3 Initiation factors (IF1, IF2, IF3)
5. Mg2+
6. GTP (guanosine triphosphate)

Step 2-Initiation-steps (e.g., prokaryotes):
1. 30S ribosome subunit + IFs/GTP bind to AUG start codon and Shine-Dalgarno
sequence composed of 8-12 purine-rich nucleotides upstream (e.g., AGGAGG).
2. Shine-Dalgarno sequence is complementary to 3’ 16S rRNA.
3. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote
proteins begin with fMet (later removed).
fMet = formylmethionine (Met modified by transformylase; AUG at all other
codon positions simply codes for Met)
mRNA 5’-AUG-3’start codon
tRNA 3’-UAC-5’ anti-codon
4. IF3 is removed and recycled.
5. IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA
subunit.
6. Results in a 70S initiation complex (mRNA, 70S, fMet-tRNA)

Step 2-Initiation, differences between prokaryotes and euakaryotes:
1. Initiator Met is not modified in eukaryotes (but eukaryotes possess initiator tRNAs).
2. No Shine-Dalgarno sequence; but rather initiation factor (IF-4F) binds to the 5’-cap on
the mature mRNA.
3. Eukaryote AUG codon is embedded in a short initiation sequence called the Kozak
sequence.
4. Eukaryote poly-A tail stimulates translation by interacting with the 5’-cap/IF-4F,
forming an mRNA circle; this is facilitated by poly-A binding protein (PABP).

Step 3-Elongation of a polypeptide:
1. Binding of the aminoacyl tRNA (charged tRNA) to the ribosome.
2. Formation of the peptide bond.
3. Translocation of the ribosome to the next codon.

3-1. Binding of the aminoacyl tRNA to the ribosome.
• Ribosomes have two sites, P site (5’) and A site (3’) relative to the mRNA.
• Synthesis begins with fMet (prokaryotes) in the P site, and aa-tRNA hydrogen
bonded to the AUG initiation codon.
• Next codon to be translated (downstream) is in the A site.
• Incoming aminoacyl-tRNA (aa-tRNA) bound to elongation factor EF-Tu + GTP
binds to the A site.
• Hydrolysis of GTP releases EF-Tu, which is recycled.
• Another elongation factor, EF-Ts, removes GDP, and binds another EF-Tu + GTP to
the next aa-tRNA.
• Cycle repeats after peptide bond and translocation.

3-2. Formation of the peptide bond.
• Two aminoacyl-tRNAs positioned in the ribosome, one in the P site (5’) and
another in the A site (3’).
• Bond is cleaved between amino acid and tRNA in the P site.
• Peptidyl transferase (catalytic RNA molecule - ribozyme) forms a peptide bond
between the free amino acid in the P site and aminoacyl-tRNA in the A site.
• tRNA in the A site now has the growing polypeptide attached to it (peptidyl-
tRNA).
Fig. 6.18

3-3. Translocation of the ribosome to the next codon.
• Final step of the elongation cycle.
• Ribosome advances one codon on the mRNA using EF-G (prokaryotes) or EF-2
(eukaryotes) and GTP.
• Binding of a charged tRNA in A site (3’) is blocked.
• Uncharged tRNA in P site (5’) is released.
• Peptidyl tRNA moves from A site to the P site.
• Vacant A site now contains a new codon.
• Charged tRNA anti-codon binds the A site, and the process is repeated until a
stop codon is encountered.
• Numbers and types of EFs differ between prokaryotes and eukaryotes.
• 8-10 ribosomes (polyribosome) simultaneously translate mRNA.

Step 4-Termination of translation:
1. Signaled by a stop codon (UAA, UAG, UGA).
2. Stop codons have no corresponding tRNA.
3. Release factors (RFs) bind to stop codon and assist the ribosome in terminating
translation.
1. RF1 recognizes UAA and UAG
2. RF2 recognizes UAA and UGA
3. RF3 stimulates termination
4. 4 termination events are triggered by release factors:
1. Peptidyl transferase (same enzyme that forms peptide bond) releases
polypeptide from the P site.
2. tRNA is released.
3. Ribosomal subunits and RF separate from mRNA.
4. fMet or Met usually is cleaved from the polypeptide.

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