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Molecular Biology Presentation
Molecular Biology Presentation
5’ Identical
base
3’ sequences
3’ 5’ 3’
5’
– 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
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
DNA
14
N 15
N
Generation
15
N 0
3
Interpreting the Data
• Energy for this reaction is derived from the release of two of the
three phosphates of the dNTP.
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 Principle replication
polymerase polymerase
(SOS inducible)
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
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
5’
3’
5’
Able to
covalently link
together 3’
3’
5’
er
i m
Pr
5’
3’
Figure 11.10
Innermost
phosphate
11-30
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
Direction of synthesis
on leading strand
3’
5’
3’
Dir 5’
e
on ction 3’
la g o
gin f syn
gs t
tra hesis 5’
nd
Direction of synthesis
of leading strand
Direction of synthesis
Of lagging strand
• 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
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
5’ SSB Proteins
Okazaki Fragments
1 ATP
Polymerase III 2
Helicase
Lagging strand 3 +
Initiator Proteins
3’
primase base pairs
Polymerase III 5’
3’
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
Fork movement
Lagging strand
Leading strand
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
The binding-
polymerization- Step 2 = Polymerization
translocation cycle can
occurs many times
The complementary
strand is made by primase,
DNA polymerase and ligase
ss Break
AP site
Covalent X-linking
Thymidine dimer
General repair mechanisms needed
• EITHER Reverse damage (e.g. PHOTOREACTIVATION)
• OR excise DNA and patch repair the region
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
-
Repair
UV
DNA Damage
WT
Repair
T-Phage suspension
Plaques !
Excision repair........
UvrABC Pol1
5’ 3’
3’ 5’
OR
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
3’ 5’ 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.
PO din
PO lexA PO recA
PO din
PO din
PO din
PO din
Low level expression
PO lexA PO recA
PO din
PO din
PO din
PO din
PO din
UV dose Dimers/
Genotype Phenotype µJ/mm2 genome
WT WT 5.0 3200
uvrA No excision repair 0.8 50
• An operon is a group of
genes that are
transcribed at the same
time.
• They usually control an
important biochemical
process. Jacob, Monod & Lwoff
• They are only found in © NobelPrize.org
prokaryotes.
Repressor RNA
protein Blocked polymerase
DNA
I
O z y a
Regulator Operator
lac operon
gene site
© 2007 Paul Billiet ODWS
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
DNA
I O z y a
© 2007 Paul Billiet ODWS
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
DNA
I O z y a
Promotor site
© 2007 Paul Billiet ODWS
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.
Repressor protein
removed
RNA polymerase
DNA
I O z y a
Promotor site
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
Activator
protein steadies
the RNA
polymerase
Transcription
DNA
I O z y a
Promotor site
© 2007 Paul Billiet ODWS
Summary
Carbohydrates Activator Repressor RNA lac Operon
protein protein polymerase
X
NEGATIVE REGULATION
X
NEGATIVE REGULATION
INDUCIBLE TRANSCRIPTION
The lac Operon
REPRESSOR
X
INDUCER
INACTIVE
REPRESSOR
X
mRNA 5’ 3’
mRNA 5’ 3’
Proteins
REPRESSOR
X
INDUCER
INACTIVE
REPRESSOR X
OPERATOR
X NO mRNA
lac repressor
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
X NO mRNA
lactose
X NO mRNA
mRNA
The RNA polymerase can now bind to the promoter and initiate
transcription of the genes. The proteins made metabolize the lactose.
NEGATIVE REGULATION
X
NEGATIVE REGULATION
REPRESSIBLE TRANSCRIPTION
Aporepressor
Co-repressor
Active repressor
X
THE trp OPERON
operator
NEGATIVE REGULATION
REPRESSIBLE TRANSCRIPTION
THE trp OPERON
Aporepressor
Operator
Co-repressor
Active repressor
X
THE trp OPERON
Aporepressor
Operator
Co-repressor
Active repressor
-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.
Transcriptional Activator
Enhancer
Transcriptional Activators
- Are proteins that activate transcription (No Dah!).
- Bind to enhancers (in eukaryotes).
- Their activity is modulated by environmental conditions.
Catabolic Enzyme
Repressor
Operon
Inducer
The lac repressor dissociates from the operator
sequence upon IPTG (inducer) binding
Cis versus Trans
Cis versus Trans
Dominant versus Recessive
Operator Mutations Reduce Repressor
Binding
The lac repressor tetramer binds two operators
Fig. 7.12
The lac operon is also regulated by glucose levels
Glucose
is
Sweeter
Co-repressor
The trp operon attenuator
No termination
Fig. 7.30
Reading: MVA pp. 760-767 and 1011-1018
Problem Ch 26: 9
N
How to Start the Feedback Loop
CI
CI/CIII
Feedback Loop is Maintained
Lamda lives Happily Thereafter…
CI
Lysogeny versus Lysis
CI wins -
Lysogeny
Cro wins -
Lytic
What If Stock Market Turns South ?
SOS
RecA+
Lambda
Protease
DNA-Protein
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
RNA PROTEIN
DNA RNA
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
XRN
DCP G
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!
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
Trans-acting element
TFB
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
Fig. 6.5
Fig. 6.6 - Three nearby insertions (+) restore the reading frame, giving normal or
near-normal function.
How was the genetic code deciphered?
2. Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA, AAC, AAA) composed of
two different bases:
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:
Degeneracy of the code is such that wobble always results in translation of the
same amino acid.
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 SER TYR CYS
PHE SER TYR CYS
LEU SER STOP STOP
LEU SER STOP TRP
LEU
LEU
LEU
LEU PRO HIS ARG
PRO HIS ARG
PRO GLN ARG
PRO GLN ARG
ILE THR ASN SER
ILE THR ASN SER
ILE THR LYS ARG
MET THR LYS ARG
VAL ALA ASP GLY
VAL ALA ASP GLY
PHE SER TYR CYS
PHE SER TYR CYS
LEU SER STOP STOP
NEUTRAL-NONPOLAR NEUTRAL-POLAR
BASIC
TRP
LEU
LEU
LEU
LEU PRO HIS ARG
PRO HIS ARG
PRO GLN ARG
PRO GLN ARG
ACIDIC
ILE THR ASN SER
ILE THR ASN SER
ILE THR LYS ARG
MET THR LYS ARG
VAL ALA ASP GLY
VAL ALA ASP GLY
Evolution of the genetic code:
Each codon possesses an inherent set of possible 1-step amino acid changes
precluding all others.
Phe, Leu, Ile, Met, Val (16 codons with T at 2nd pos.) possess 104 possible
evolutionary pathways.
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).
4. Amino acids bound to tRNAs are transported to the ribosome. Facilitated by:
Complementary base-pairing between the mRNA codon and the tRNA anti-
codon.
1. Charging of tRNA
2. Initiation
3. Elongation (3 steps)
4. Termination
Step 1-Charging of tRNA (aminoacylation)
5. Sequence of events:
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).
3. Initiator tRNA (fMet tRNA) binds AUG (with 30S subunit). All new prokaryote
proteins begin with fMet (later removed).
5. IF1 & IF2 are released and GTP is hydrolysed, catalyzing the binding of 50S rRNA
subunit.
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.
• 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.
• Another elongation factor, EF-Ts, removes GDP, and binds another EF-Tu + GTP to
the next aa-tRNA.
• Two aminoacyl-tRNAs positioned in the ribosome, one in the P site (5’) and
another in the A site (3’).
• 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.
• Ribosome advances one codon on the mRNA using EF-G (prokaryotes) or EF-2
(eukaryotes) and GTP.
• Charged tRNA anti-codon binds the A site, and the process is repeated until a stop
codon is encountered.
3. Release factors (RFs) bind to stop codon and assist the ribosome in terminating
translation.
2. tRNA is released.