Semester 1 Flow of Genetic Information Flashcards
What is the genetic material?
Nucleic acids are the genetic material
What is cell division called in different types of cells?
In unicellular life its called reproduction
In multicellular life its called growth
Creating new cells means synthesising new DNA
What is binary fission?
The doubling of DNA
2 –> 4 –> 8 –> 16
This is the most common type of cell division but doesn’t occur in yeast
What are some generic genome sizes?
NON CELLULAR:
- Bacteriophage = 5380
- Mitochondrial = 16,569
CELLULAR:
- E.Coli = 4.6x10^6
- Fruit Fly = 1.8x10^8
- Mouse = 2.7x10^9
- Human = 3.1x10^9
Exceptions are present;
- Amoeba Dubia = 6.7x10^11
Even though it is non cellular
DNA polynucleotide chain composition?
A polynucleotide DNA chain is composed of three components:
- Nitrogenous base
- Pentose sugar (deoxyribose in DNA)
- Phosphate group.
There are two types of nitrogenous bases:
- Purines (adenine and guanine)
- Pyrimidines (cytosine and thymine)
A nitrogenous base is connected to the 1′ carbon of the pentose sugar through an N-glycosidic linkage to form a nucleoside such as:
- Adenosine
- Guanosine
- Cytidine
- Thymidine
What is the structure of DNA?
Double-helix
Right-handed
Antiparallel (one strand runs in opposite direction of the other, 5’ -> 3’, 3’ -> 5’)
Phosphodiester backbone
~10 nucleotides per turn
Bases are on the inside
Hydrogen bonds between bases on opposite strands
What is complementary base-pairing?
The idea that Guanine and Cytosine will always bond together and Adenine and Thymine will always bond together
G – C = 3 Hydrogen bonds
(stronger bond, more stable)
A – T = 2 Hydrogen bonds
(weaker bond, less stable)
Chargaffs rule states that the amount of A and T, and the amount of G and C will always be the same as each other
What is required for DNA synthesis?
In lab:
- Taq (DNA Polymerase)
- dNTPs
- Template DNA
- Primers
In cell:
- DNA Polymerase III
- dNTPs
- Template DNA
- Primers
- But also… many many other proteins are involved in a cell
What are the types of DNA Polymerases and what do they do?
Named in order of discovery not importance
Pol I:
- DNA repair and replication
Pol II:
- DNA repair
Pol III:
- Principal DNA replication enzyme
Pol IV:
- DNA repair
Pol V:
- DNA repair
All synthesize 5’ –> 3’
Comparison of Pol I and Pol III?
Pol 1:
- One gene
- 109kDa
- 400 copies per cell
- 10 nucleotides/s
- 20-100 nucleotides at a time (before falling off)
- 100 hours per genome
Pol III:
- 22 genes
- 10^6kDa
- 10 copies per cell
- 1600 nucleotides/s
- >50,000 nucleotides at a time
- 40 minutes per genome
How can we determine which genes (proteins) are important in DNA replication?
Simple knock-outs will be lethal
Temperature-sensitive mutants allow proteins to be switched on or off by changing the temperature
- e.g. protein works at 20, but not 37°C
Allow cells to begin replication, then deactivate one protein to see the effect
- Quick stop mutants: Replication immediately stops
- Slow stop mutants: Current round of replication finishes, but a new one can’t start
What are some issues that arise with DNA replication?
Strands being coiled (topology)
Circular DNA molecules (topology)
Antiparallel strands (polarity & topology)
Mutations/errors (fidelity)
What is the issue with strands being coiled and what is the solution?
PROBLEM:
- DNA strands are plectonemically coiled so they will have to be unwound to separate them without getting them tangled
RESOLUTION:
- Helicases can separate and unwind the duplex using ATP hydrolysis
What are helicases and what is their structure??
Helicases separate and unwind doulbe stranded DNA using ATP hydrolysis (3 bp/ATP)
Hexamer ring surrounds a single DNA strand
Helicase Function & Action
Conformational changes in the helicase pull on the DNA strand, separating it from its partner
Helicases move towards the 3’ end of the strand they are clamped to
One helicase on each strand of DNA
Helicases separate and unwind the two DNA strands, creating a replication bubble
Uncoiling at one part of the duplex, creates mechanical strain in the rest of the molecule
DNA Replication Speed & Mechanics
Pol III can synthesize ~1600 nucleotides/s
There are ~10 nucleotides per turn
Helicases must rotate DNA at ~10,000rpm!
What is an issue with circular chromosomes?
When DNA helicases unwind and seperate DNA, it can create torsional strain elsewhere in the duplex which results in supercoiling
What equation can DNA topology be described by?
Lk = T + W
Lk = linking number (fixed value in circular DNA, number of times the DNA crosses over itself)
T = twist - number of turns of the duplex
Twist = N/h (number of BP / helical repeats)
N cant change but h can (over/under-winding)
W = writhe - number of duplex self crossings
The relaxed form, where W=0, is called Lk0
If Lk > Lk0 then there is +ve supercoiling (+W)
If Lk < Lk0 then there is –ve supercoiling (-W)
Different amount of writhe results?
What is the equation used to determine superhelical density?
This is a normalised way of expressing how supercoiled a piece of DNA is, removing the effect of chain length.
In relaxed DNA, σ = 0
Sign indicates type of supercoiling
What is the biological significance of negatively supercoiled DNA?
Purified cellular DNA is always slightly negatively supercoiled
σ = -0.06 (eukaryotes and prokaryotes)
Conversion of –ve Writhe to less Twist aids unwinding for transcription and replication
Allows easier separation of DNA
Also, Eukaryotic DNA is negatively supercoiled around histones when forming a nucleosome
What is the biological significance of positively supercoiled DNA?
Helicase-based unwinding results in overwinding elsewhere so will always produce positive supercoiling
Overwinding will resist replication fork movement
Lk can’t change to relieve the stress without breaking the phosphodiester bonds
So… Positive supercoils form
Also applies to very long linear DNA
What is the result of positive supercoiling?
Positive supercoiling prevents DNA replication
It will need to be removed for replication to continue
How can we remove or add supercoiling?
Specific enzymes that introduce / remove supercoils
TOPOISOMERASES:
Type I:
- Cleave backbone of one strand, allowing duplex rotation and loss of negative supercoils
Type II:
- Cleave backbone of both strands, using ATP and introduces a negative supercoil
DNA backbone is sealed after manipulation
Supercoil removal via Topoisomerase Type I mechanism
Phosphodiester bond is transferred to Tyr residue on enzyme, breaking one DNA strand
The unbroken strand is passed through the gap
The phosphodiester bond is transferred back to DNA, reforming the backbone on the other side
Supercoil removal via Topoisomerase Type II mechanism
What is special about supercoiled dna in gel?
Supercoiled DNA runs faster than linear DNA in an agarose gel
What issues occur when chromosomes are circular
DNA replication is bidirectional, so with circular DNA the DNA Polymerases can collide with each other
Also, as the DNA is seperated and expanded, two duplexes will be created that when finished, will be
entwined with one another
When circular DNA molecules are replicated, the two daughter rings are interlocked and form a CATANENE
How are catanenes dealt with?
Topoisomerase IV can be used
Cleaves 1 duplex, the second duplex passes through and then the cleavage is sealed
Process called decatenation
How do antiparallel DNA strands manage synchronous replication?
DNA strands are antiparallel: one 5’→3’ and the other 3’→5’
DNA polymerase can only add nucleotides in the 5’→3’ direction
The leading strand is continuously synthesized in the direction of the replication fork
The lagging strand is synthesized discontinuously in short segments called Okazaki fragments
To facilitate this, the lagging strand loops out, forming a structure that allows the DNA polymerase to synthesize the strand in the 5’→3’ direction, but effectively in the opposite direction of replication fork movement
What is the end result of antiparallel strand replication?
End result of semi-discontinuous replication is two, non-identical dsDNA molecules
Chromosome is replicated, but lagging strand copy is unfinished, with ‘nicks’ in the new backbone, because Pol III can’t join fragments
What did Okazaki investigate?
Okazaki investigated the synthesis of DNA over time using radioactivity
1) E.coli culture infected with T4 bacteriophage
2) Add 3H-TTP (tritiated thymidine) so that the new DNA strands created will be radioactive
4) Mapped out sizes of radioactive ssDNA over time using density centrifugation
5) Small new strands of radioactive DNA were always present, as pictured
6) Even after full size strand had been created, new smaller strands were still present, proving size of radioactive ssDNA doesn’t simply increase smoothly over time. Short pieces are always present
Okazaki fragment explanation?
Short fragments are repeatedly being made on the lagging strand as new sections of DNA are unwound
After a few seconds they are joined to each other and to non-radioactive pieces that had already been synthesized
Results in a size jump
Further evidence of Okazaki fragments using chase experiment
Chase/Pulse experiments carried out
After 2 seconds the 3H-TTP
is removed and “chased” with normal TTP
Radioactivity all moves down the tube as pictured
The observations supported the hypothesis that the lagging strand is synthesised discontinuously in short fragments, which are later connected to form a continuous strand.
Further evidence of Okazaki fragments using DNA Ligase mutant experiment
DNA Ligase consumes ATP to join Okazaki fragments of DNA together
When DNA Ligase mutant is used instead of DNA Ligase, the fragments can not be joined together
This results in density centrifugation only showing short, small okazaki fragments and no fully formed DNA fragments, as pictured
Are okazaki fragments completely DNA?
Not completely DNA
Primer is RNA
We know this because tiny fragments are left over after using DNAse on Okazaki fragments
What are some mutations / errors that occur during DNA synthesis?
1) Incorrect nucleotide added
2) RNA nucleotide instead of DNA nucleotide
3) Nicks in backbone (fragments)
How is adding the wrong nucleotide avoided?
The incoming dNTP has to form the
correct base pair to fit properly into the
polymerase active site
DNA Polymerase has an induced fit mechanism
Has binding and shape discrimination
Incorrect dNTP are excluded by steric collisions, even if there are hydrogen bonds possible
How often does the addition of the wrong nucleotide occur?
Pol III adds the wrong dNTP at a rate of 1 per 100,000 bp (error rate of 10-5)
Would be ~ 46 point mutations per E.coli replication
Error rate drops to 10-7 with proofreading activity of polymerases
What is proofreading in dna polymerase?
Proofreading in DNA polymerase is the enzyme’s ability to detect and correct mistakes during DNA replication
When DNA polymerase inserts an incorrect nucleotide opposite the template strand, it can recognise this error and correct it
Proofreading in Pol III?
Pol III has 3’-5’ exonuclease activity
- Can remove the last nucleotide added, if it was incorrect
A mismatched nt at the 3’ end won’t be in the right place to add the next dNTP so:
- The polymerase stalls
The end of the strand become substrate
for the exonuclease active site, which cleaves the terminal phosphodiester bond, releasing a dNMP
What are the activities in Pol I?
An enzyme with multiple activities:
- 5’-3’ polymerase (synthesis)
- 3’-5’ exonuclease (proofreading)
- 5’-3’ exonuclease (nick translation)
When pol I is treated with protease it produces 2 main fragments:
Small N-Terminal Fragment
- Contains 5’-3’ exonuclease
Large C-Terminal Fragment (Klenow Fragment)
- Contains polymerase
- Contains 3’-5’ exonuclease
DNA Pol I 5’-3’ exonuclease?
Nick translation
Binds to nick in synthesised dna fragment
Will then remove nucleotides ahead of it and replace with correct nucleotide
Doesn’t remove nick, just moves it further along
Is there RNA in ozaki fragments? What removes the RNA?
Yes, there is RNA at the 5’ end, and a nick at their 3’ end
Pol I binds to the nicks in in the fragments, but Pol III does not
RNA primers are removed by Pol I 5’-3’ exonuclease
Pol I detaches after 100bp and a new nick is left behind
Pol I starts joining together the fragments while Pol III is still expanding the strand
What is RNAse H?
It is a protein that removes RNA in DNA strands
It cant cut between RNA and DNA nucleotides, so leaves a gap between the end 2
Pol I comes along and fills the gap
What are pseudo-Okazaki fragments?
Leading strand also consists of fragments that need to be joined together
Strand is grown continuously and nicks are added in afterwards
Why are pseudo-Okazaki fragments created?
The synthetic pathway for making dTTP, includes dUTP
Enzyme 7 can sometimes add a dUTP instead of a dTTP
During synthesis, Pol III will incorporate U instead of T, 1 out every 300 times (once every 1200 nt synthesized) by accident
Since U shouldn’t be present in DNA it must be removed:
- Nicks in chain
- Fragments of ~1200nt
If enzyme 4 is mutated:
- More U & shorter fragments
Why is U in the place of T a problem?
When U is accidentally added in place of a G there is no problem
When U is formed by deamination of C, mutations are caused
Presence of U in DNA is therefore offensive as it suggest damage and therefore it must be removed
How is U removed from DNA?
Base is removed by:
- Uracil-N-Glycosylase (gene = ung)
Baseless nucleotide is recognised and phosphodiester backbone cleaved by:
- AP(apyrimidinic) endonuclease (xthA)
Results in nicked DNA with incorrect nucleotide
How are nicks in dna sealed?
DNA Ligase seals nicks in DNA
Can’t do RNA - DNA nicks (wont accidentally join in the RNA primer)
What is the origin of DNA replication?
Circular chromosomes and plasmids have a single origin of replication (ori)
The origin is a region of repetitive dsDNA that is rich in A-T
Different in different organisms/plasmids e.g. oriC in E.coli chromosome
oriC is about 245bp long, split into 2 main sections:
- 3, 13-bp repeats
- 4, 9-bp repeats
What is DnaA?
It is a protein that multiple of bind to 9bp repeats in the ori, and cause it to coil which in turn induces unwinding at the 13-bp repeats
It uses ATP to separate (melt) the duplex at the 13bp repeats
What is DnaC and DnaB helicase?
DnaC is a protein that binds to the ssDNA created by DnaA and loads a DnaB helicase onto one strand, facing the 3’ end
The DnaC then detaches and the helicase moves to fork
What is DnaG primase?
After 65 nucleotides have been unwound by the DnaB helicases, DnaG primase enzymes bind to the helicases, forming a ‘primosome’
Primosomes are the functional units that create Primase
What is the primase enzyme?
Creates a primer for DNA polymerase to bind to
Primase is an RNA polymerase (but not the one involved in transcription)
It is self priming, adding RNA 5’-3’ on a 3’-5’ DNA strand (GTA site preferred)
RNA primers are -10 nucleotides in length
It has no editing functions (no proof reading)
Activity is increased in the presence of helicase and itself (co-operativity)
Primase synthesises a 10 nucleotide RNA primer and detaches from helicase
What is single stranded binding protein? (SSB)
Single stranded binding protein (SSB) binds to exposed ssDNA preventing re-annealing
- Encoded by ssb gene
- Forms a tetramer
- Not sequence specific
- Leaves bases exposed when bound
- Binds co-operatively to ssDNA
- Therefore, proteins at the ends are bound less well and are easier to displace by polymerases
What does the primer strand and SSB trigger?
First primer and SSB trigger arrival of the Pol III holoenzyme at the 3’ end of the primer
What is the structure of Pol III holoenzyme?
How does Pol III bind to the primer?
The clamp loader on Pol III loads a beta-clamp onto the DNA
Pol III core then binds to the beta-clamp
What does the clamp loader do?
Binds β clamp proteins
Transfers the β clamp onto DNA at primer 3’ end
ATP Hydrolysis is required to detach the clamp loader from the beta-clamp
Beta-clamp structure?
Encoded by dnaN gene
Forms a ring dimer
Not sequence specific
Binds to the Pol III core and imparts processivity
Changes ability of Pol III from doing 10s of basepairs at a time to >50,000
Pol III on its own isnt any better than Pol I at staying on DNA, beta-clamp improves this
What happens as Pol III catches the helicase?
Pol III travels to replication fork, synthesising the leading strand as it goes and displacing SSB
As it catches the helicase, a replisome forms
What is a replisome?
It is a region around the replication fork that contains these proteins:
- Pol III Holoenzyme
- Primosome
It occupies around 50nm area around the replication fork
Initiation of Lagging Strand Synthesis
As helicase unwinds the duplex, primase re-binds and synthesises a new primer
A β clamp is added to the primer by the clamp loader
A Pol III core binds once enough ssDNA has emerged for the β clamp to reach it
Primer 1 bound, with correct polarity
First Okazaki Fragment Formation
First Okazaki fragment starts
DNA is pulled by helicase and Pol III
The lagging strand loops out, picking up SSB
The first Okazaki fragment is finished
Cycle Repeat for Subsequent Fragments after okazaki fragment formation
Primase re-binds helicase, then adds a second primer
Pol III core and β clamp detach from DNA, releasing completed fragment
Primer 2 gets a β clamp
Looping process repeats for primer 2
How are the lagging strands okazaki fragments edited?
Pol I binds the end of the first Okazaki fragment and replaces the RNA with DNA
DNA ligase seals the nick
Process repeats for each fragment
How are the replication forks prevented from overshooting?
Ter and Tus
To prevent the forks overshooting there are 23 bp sequences called Ter
The Ter sequences bind the Tus protein
Tus can be displaced by the fork only in the correct direction, otherwise the helicase stalls
What is topoisomerase IV and what is it used for?
As the forks get within 200bp of each other, there is no longer room for DNA gyrase to bind
This results in positive supercoiling, which is relieved by
topoisomerase IV decatenating the molecules
The topoisomerase IV binds to one of the strands of dsDNA, cuts it, passes the other dsDNA strand through it, and then rejoins the original strand
How does initiation only occur once per cell cycle?
OriC contains GATC sequences (within the 13bp repeats), which are substrates (binding sites) for dam (DNA adenosine methylase)
The ‘A’ within the GATC sequences in the 13bp repeats will have a methyl group attached to it
dam methylates N6 of adenosine
After replication, only one strand will be methylated (template strand) (referred to as hemimethylated, one strand methylated, one strand not methylated)
Hemimethylated GATC sequence bind SeqA protein
What does the SeqA protein do?
Prevents DnaA binding to OriC
The GATC sites are methylated by dam very slowly (~13 mins)
So newly synthesized dsDNA remains hemimethylated and new initiation is prevented
SeqA also binds DNA to the membrane, localising it to the membrane (assists with separating two chromosomes during cell division)
What are the similarities in eukaryotic and prokaryotic DNA replication?
Both use helicases to unwind the duplex and create replication forks
Both use SSBs to hold the ssDNA apart
Both use RNA primers for the polymerase/clamp
Both use various DNA polymerases for synthesis of the new DNA on leading and lagging strands
What are the differences in eukaryotic and prokaryotic DNA replication?
Eukaryotic occurs in the nucleus not the cytoplasm
Eukaryotes have much more genetic material to replicate (can be 4 orders of magnitude greater)
More than 1 chromosome in eukaryotes
Linear chromosomes (except mitochondria) in eukaryotes
Eukaryotes have more packaging (nucleosomes, histones)
Eukaryotic replication has multiple origin points (10s-1000s), replication starts from multiple points
DNA polymerases are much slower in eukaryotes (50nt/s vs 1000nt/s)
Polymerases synthesising the leading and lagging strands aren’t physically linked together in eukaryotes
Eukaryotes do NOT have any polymerases with a 5’-3’ exonuclease
Okazaki fragments are much shorter in eukaryotes (165nt vs 2000nt)
DNA replication initiation in eukaryotes
Tightly linked to the cell cycle
Initiation must only happen once per cycle
A two step process ensures this ^
What binds to autonomously replicating sequences?
In yeast, an Origin Recognition Complex of proteins (ORC) binds to the A region of the ARS
It is unclear how this works in higher eukaryotes
What occurs after ORC binds to the ARS?
Accessory proteins (licensing factors) accumulate in the nucleus during G1
Cdc6 and Cdt1 (examples of licensing factors) bind to ORC
What happens after licensing factors bind the ORC?
Two helicases are loaded by the licensing factors
The licensing factors then leave
Pre-replication complex is formed
Is every pre-replication complex used every time?
Not every pre-replication complex is used in each round
of replication
Replication forks from one origin will pass through another origin (passive replication)
How are RNA primers removed in eukaryotic dna replication?
Pol δ & Pol ε displace the primers and push them to the side generating an RNA ‘flap’
The RNA “flap” produced by Pol δ or ε can be mostly digested by RNAse H1, with 1 RNA nucleotide left at the end
Flap 1 endonuclease (FEN1) binds PCNA and can remove any incorrect nt, by cutting off a section of ssDNA/RNA
DNA ligase fills in nick generated by FEN1
What is the end replication problem in linear chromosomes?
Affects the lagging strand
Last RNA primer may not be at the extreme 3’ end of the DNA which results in a missed section
So how do embryonal cells, cancer cells, stem cells
and other types divide more times than the Hayflick limit?
They can synthesise new telomeric DNA
Telomerase is a ribonucleoprotein
It contains an RNA strand (450 nt) which has the sequence CUAACCUAAC
This acts as a template for the synthesis of new telomeric repeats, growing the telomere
Telomerase mechanism with Pol alpha nad Pol Delta
How does bacteriophage DNA replication occur?
They have a small circular genome
Uses a ‘rolling circle’ mechanism to continuously synthesise new DNA
Called Sigma (σ) replication
- Start off with circular DNA molecule
- Nick is deliberately created in DNA
- Nick acts as binding site for polymerase
- Pol displaces existing strand and replaces with newly synthesised one
What is DNA damage?
Something wrong with DNA that may lead to:
- Mutations
- Unable to replicate
What are replication errors?
Normal replication introduces the wrong base once every 10^7 bp, even with proofreading
There is a good chance this error will be repaired
Repair systems reduce error rate to 10^-10
Some repetitive regions cause slippage of the growing strand insertion of more repeats which can’t be repaired
Slippage is particularly prevalent with trinucleotide repeats
What are tautomerisation errors in dna replication?
Tautomerization is a reversible chemical process in which a DNA base temporarily changes its structure, leading to the incorrect pairing of nucleotides during DNA replication
In the example, the shifted enol form of T binds G instead of A
The mutations are consolidated on next replication
What are deamination errors in dna replication?
It is the loss of a base amino group
C –> Uracil (pairs A)
A –> Inosine/Hypoxanthine (pairs C)
G –> Xanthine (pairs C less strongly)
C, A and G deaminations can be repaired
- Around 100 C are converted to U per day
- Around 1 a day per cell for A and G
What are depurination errors in dna replication?
Occurs mainly in A or G
It is the cleavage of base-sugar bond
Forms an abasic site (apurinic)
Around 10,000 purine glycosidic bonds
hydrolyse/cell/day
Around 600 pyrimidine glycosidic bonds
hydrolyse/cell/day
4x more likely in ssDNA
Mutation is consolidated on next replication
What are intercalating agents?
They are chemicals that insert themselves between bases, and distort the DNA
They look similar to bases, so are able to insert themselves
e.g. ethidium bromide
Intercalating agents cause frameshift mutations
- Insertions
- Deletions
What are base analogues?
These are chemicals that are very similar in structure to bases
For example, bromouracil (T analogue) (pictured)
Because the structure is so similar, they are incorporated into the DNA
These base analogues are more prone to tautomeric shifts
What are alkylating agents?
Alkylating agents add carbon groups (alkyl) to nucleotides
e.g:
- Nitrosamines
- Methyl bromide
Most common is G –> O6-Methyl-G, which is G with an additional methyl group
O6-Methyl-G pairs T
Alkylation can also speed up depurination
How does UV light cause DNA errors?
It can cause UV-induced formation of dimers between adjacent thymines
Stops correct base-pairing in polymerases
How does direct reversal of demethylation occur?
Occurs via specific reversal proteins
e.g. O6-methylguanine-DNA methyltransferase (MGMT)
Transfers methyl/ethyl group from G to a Cys
residue on itself:
→ G restored
How does direct reversal of dimers created via ultraviolet light occur?
Via DNA photolyase
Absorbs blue light and breaks T-T internucleotide bonds, using FADH → 2 Ts restored
Mammals have to use another system for repairing thymine dimers,as they don’t have this enzyme
How does UDGase discriminate between U and T?
T has a methyl group and the U doesn’t
That methyl group clashes with the tyrisine residue on the active site
There is a separate TDGase for GT pairs
If Tyr is mutated to Ala, both T and U will be removed by the enzyme
What is nucleotide excision repair?
Removal of oligonucleotide fragments from one strand (bulk correction)
Triggered by changes in the physical structure of the duplex as a result of damage
Corrects any of the types of sequence damage e.g. thymine dimer removal
Achieved by a protein complex called UvrABC exinuclease in E.coli (many more proteins in eukaryotes)
How does methyl-directed mismatch repair work?
Hemi-methylation provides the information on which strand is parent (correct) and daughter
- MutH binds to unmethylated GATC at OriC,
identifying the daughter strand. - MutS binds to a distorted site on the duplex
- MutL binds to MutS
- MutL/MutS complex travels back to the origin and activates MutH
- MutH cleaves daughter strand (nicked)
- Specialized helicase and exonucleases remove nt until past the distortion
- Pol III fills in missing nt. DNA ligase seals nick
Where does replication, transcription and translation occur in prokaryotes?
Replication + transcription + translation all happens in the cytoplasm in prokaryotes
What are all of the types of transcription control?
- Transcription control
Regulation of transcription is the most common form of control of protein production
- RNA processing control
- RNA transport and localization control
- Translation control
- mRNA degradation control
- Protein activity control
(Long non-coding RNA)
- (>200 nt, not transcribed. Involved in regulation through binding RNA, proteins and DNA thus influencing important interactions)
DNA transcription labelling
What does RNA polymerase do?
Separates the 2 strands of DNA
Uses one of the DNA strands as a template for RNA synthesis
Does not require a primer
It is very accurate 1/10,000 bases
Moves along the gene in a 5’ to 3’ direction
Synthesises a complementary RNA copy of the DNA template strand
How many RNA copies are made per gene?
Many RNA copies are made at the same time
Multiple RNA polymerases per gene
Multiple transcripts per gene
- As soon as the first RNA polymerase starts to move down the gene, the next polymerase can bind and initiate RNA transcription
What is the mechanism of transcription?
RNA Polymerase binds, has helicase activity to begin with, breaking hydrogen bonds and unwinding DNA
Does this as it moves in 5’ to 3’ direction, forming a transcription bubble, which opens up so that the template strand can be copied
Free nucleotides join and are added to complement the template strand and generate a copy of the coding strand
Length of the bubble is 12-14bp
Reaction rate approx 40 bases per sec
What is an operon?
Single unit that controls multiple genes
Genes encoding for proteins in the same pathway are located adjacent to one other and controlled as a single unit that is transcribed into a polycistronic RNA - No introns
What is the lac operon?
Deals with changes in lactose
Switches on genes that encode enzymes which are needed to metabolise that sugar
LacZ –> LacY –> LacA
Contains three genes:
- B-Galactosidase
- Permease
- Transacetylase
What are bacterial promoters?
Control of transcription most commonly occurs at transcription initiation
Promoters are the sites of transcription initiation in the DNA
Promoters are recognised by RNA polymerase by having a consensus (common pattern of) DNA sequence
The first is a hexamer (6bp) at -35
Second is a TATAAT sequence at -10 (Pribnow box)
- Asymmetric (only in 1 DNA strand), hence RNA polymerase knows which way to go!
What are alternative sigma factors?
e.g., Heat Shock
σ32 is induced by high temperatures
σ32 is induced by the accumulation of unfolded proteins
σ32 recognises a different -35 and -10 sequence
What are the stages of the bacterial growth curve?
Lag Phase
- No increase in number of living cells
Log phase
- Exponential increase in number of living bacterial cells
Stationary phase
- Plateau in number of living cells; rate of cell division and death roughly equal
Death or decline phase
- Exponential decrease in number of living bacterial cells
sigma70 in log phase
sigma38 replaces it near stationary phase
Initiation in bacteria
RNA polymerase binds to the promoter
Unwinds the DNA
Transcription begins
Regulates transcription
Elongation in bacteria
RNA polymerase makes an RNA copy complementary to the template strand
Bacterial RNA polymerase: only 1, with 5 units (sigma)
Lac Operon regulation
Regulated by both negative and positive regulators
Negative = lac repressor (lacI)
Positive = Catabolite activating protein (CAP)
LacI, the lac repressor, is said to be constitutively expressed hence it is always on hence lacI is always present
The lacI gene encodes the lac repressor
The lac repressor is the protein that binds down stream and its job is to control transcription of the lactose operon
What happens to the lac repressor when lactose is absent?
Lac repressor is produced
Lac repressor binds to the operator
Lac operon transcription blocked
What happens to the lac repressor when lactose is present?
Allolactose (derived from lactose metabolism) is produced
Allolactose binds to the lac repressor
This induces a conformational change in LacI
Repressor cannot bind to operator
How does lac repressor regulate
Lac repressor binds to the operator site which overlaps the start site of transcription
The sequence where lac repressor binds and the first A is where transcription starts and this is labelled plus 1
Lac repressor binding to operator sites
Lac repressor binds to the operator site which overlaps the start site of transcription
The lac repressor must bind 2 out of 3 sites
- Repressor can bind to O1 and O2 or O1 and O3
- But not to O2 and O3
CAP function
When glucose is available it is used in preference to other sugars (e.g., lactose)
Low Glucose –> cAMP High –> CAP Active
High Glucose –> cAMP Low –> CAP Inactive
When cAMP is high it binds to CAP and causes conformational change allowing it to bind upstream of the -35 sequence and stabilizes RNA polymerase
Rho dependant termination
Rho is a prokaryotic transcription protein
~275kD hexamer
Each subunit has a RNA binding domain & an ATP hydrolysis domain: moves along the RNA
Also requires an inverted repeat
Stalls the RNA polymerase
Rho is a helicase
Unwinds DNA:RNA hybrids
What is the Typical arrangement of a eukaryotic promoter region
Core promoter elements:
- TATA Box (-25)
- Inr (+1)
- DPE (+25)
Regulatory elements:
Proximal
- CAAT Box
- GC Box
Distal
- Enhancer Elements
Typical arrangement of a TATA core promoter
TATA Box and INR (initiator) make up the ‘Core Promoter Region’
INR:
- Simplest functional promoter
- Can initiate basal transcription in absence of TATA box
- Conserved Y is pyrimidine
- Consensus sequence YYANWYY in humans, where, Y = C/T, W = A/ T, N=A/C/G/T, and +1 is underlined.
- The INR element facilitates binding of TFIID
TATA Box:
- Conserved sequence TATAAAA
- Approx 25 bp upstream of initiator
- Found in ~30% of promoters
- Core promoter similar concept in Pribnow box (prokaryotes) and TATA box (eukaryotes).
What is an RNA Pol II Pre initiation complex?
Eukaryotic RNA Pol II does not bind directly to the TATA box region of the DNA, it requires proteins called “General Transcription Factors (GTF)” to position it
Each transcription factor is a complex of polypetides
TFIID begins whole process
Need to assemble an RNApol II pre-initiation complex
This positions the RNA pol II over transcription start sites.
How is the RNA Pol II Pre initiation complex formed?
General transcription factors for RNA Pol II (TFII) bind to the TATA box
The first transcription factor added to the TATA box is TFIID
The TFIID complex consists of TBP (TATA binding protein) and TAF (TATA associated factors) and acts as a scaffold for remainder of the preinitiation complex
Next, TFIIA binds followed by TFIIB
TFIIB is able to then recruit RNA Pol II to the TATA Box
TFIIF accompanies the RNAPol II and stabilises it
TFIIE and TFIIH bind and make up the initiation complex
Pre- initiation complex (PIC) is now assembled
What is TFIIH and TFIIE Function?
The function of TFIIH is a helicase, it melts the DNA and separates the strands
A short sequence of pre-mRNA is added to the gap
TFIIH and TFIIE also have kinase activity
Phosphorylation of the C terminal domain of RNApol II by TFIIH then occurs
After phosphorylation, TFs are released from the complex
The phosphorylation also causes a conformational change in RNApol II, which tightens the grip it has, also allows binding of new factors (elongation factors) that increase efficiency
What determines cell fate?
Cell fate is determined by:
- Gene expression
- Cell-cell/cell-matrix interactions
- External factors e.g. hormones
WHICH CAUSES:
- Unique transcriptional factor (TFs) environment for each cell type
What are upstream sequence elements (USE)?
Transcription can be enhanced by the binding of transcription factors to sites upstream of the PIC
Upstream sequence elements (USE)
Examples:
GC Box:
- GGGCGG
- Binds SP1 TF
CAAT Box:
- GG(T/C)CAATCT
- Binds CAAT box TF
These must be in same orientation as the RNA pol II initiation site
Upstream position is important.
What are enhancers (eukaryotes)
Regulatory sequences that act at a distance
Cis acting (up to 1Mb away) with reversible orientation
Bound by activator proteins
These interact with the mediator complex
Encourage binding of RNApol II
Enhancers work because DNA structure is complex and twisted around itself, so regions distant in sequence may be physically close to each other
What are some properties of enhancer elements?
- They can activate transcription when placed thousands of bp away from the TATA box
- They act in either orientation
- Can act when placed upstream or downstream of the TATA box, or when placed within an intron
What are transcription factors structure
TFs are proteins made up of amino acids hence their 3D structure is important to their function
Modular structure:
- One region binds DNA
- All the amino acids for DNA binding in 1 region
- Another region binds to other components
- Also contains activation or inhibitory domains
What are zinc fingers?
Contains a loop of 23 aa
Usually have multiple zinc fingers per TF
The linker between the fingers is 7-8 aa
a-helix contacts the major groove of DNA
Often multiple zinc fingers involved in binding the specific DNA sequence.
Zn2+ ion does not directly interact with the DNA but is essential for the folding of the finger.
Zinc fingers bind both to the major and minor grooves.
What is a helix turn helix
Two helices held at a fixed angle
Recognition helix binds major groove of DNA
Bind DNA as dimers, so the 2 recognition helices are separated by one turn of the DNA helix
