L5 - Desire For DNA Flashcards
Why is there a desire for DNA?
Average protein of 500 amino acids requires an mRNA with an open reading frame with 1503 nucleotides (3 bases for each AA = 500x3= 1500) + 3 bases because these 3 are the the ones that make up the stop codon
In a protein world that only has 1000 proteins (not many) this means you require 1503 nucleotides/1 protein so need 1503x1000= 1.5 million nucleotides in the open reading frames (+ need more nucleotides that aren’t in the ORFs)
So desire for long stable sequences so the protein world can be sustained. Major issue = RNA NOT THAT STABLE
Why is RNA not that stable?
RNA spontaneously mutates; spontaneous deamination of cytosine into uracil
1/16000 cytosines per day
What is the issue with the cytosine in RNA spontaneously deaminating to uracil?
RNA cannot distinguish uracil from cytosine
So Cytosine will slowly deaminate to uracil and because RNA cannot tell the difference and therefore change them back to cytosine, over time you get RNA that no longer has cytosines
What makes RNA sequences unstable over many generations?
Deamination of cytosine to uracil
This mutation is never repaired and it stays as uracil
(Important reason why DNA was needed)
2nd reason DNA is better
RNA has 2’ OH
Makes H bonds and allows RNA to bond
Little reactive so can be modified so causes instability of RNA
RNA vs DNA
Two chemical difference:
2’OH is absent in DNA ( no 2’ OH on deoxyribose, therefore also DNA doenst fold like RNA does)
DNA has thymine, RNA has uracil. Thymine has an extra methyl group at certain position (see slide) whereas uracil doesn’t.
This is a trick = deaminated cytosines turn to uracil,
What’s special about DNA (deamination)
Has the ability to recognise where the deamination of a cytosine to a uracil has occurred.
(If there’s a uracil in DNA it must have come from a cytosine because other than that DNA should have uracil’s)
Makes DNA more stabke
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Two H bonds
A and T
Three H bonds
Three ah bonds
G C
What’s good about DNA not have 2’OH
It means it only folds as a double helix with its reverse complements
Base pairing between two strands
This means if there’s a mutation in one base, the other base in the other strand can be used to correct the sequence
Double helix = more stability
How to make a reverse complement of DNA
Anti parallèle strands
Turn the sequence 180 degrees
Then write out the complements
DNA double helix
Nitrogenous bases (slightly positively charged because it’s a base and can accept protons)
Phosphate backbone (negatively charged)
Major groove - allows other molecules to bind to DNA and recognise specific bases (eg. Proteins or TFs)
Minor groove -
In nature we only have what type of DNA helix
Right handed
How much DNA does each cell have
2.2 metres
dNTP
Deoxynucleotides
N = a base
Tri phosphate, deoxyribose, base
How are dNTPs synthesised?
RNR (ribonucleoside reductase) takes a nucleoside diphosphate and removes the 2’OH from the ribose sugar - produces dNDP
Kinase enzyme adds phosphate to dNDP. Produces dNTP
What does RNR make
dADP
dGDP
dCDP
dUDP
In DNA synthesis why does RNR make dUDP even though DNA doesn’t have any uracil’s in it?
dTDP is made from dUDP
So dUDP is made by RNR because even though U is an RNA base and here DNA is being made, it has to be made so dTDP can be made from it
What is a DNA relic in DNA synthesis ?
The fact that dTDP has to be made from dUDP - indirect synthesis of thymine from uracil - example DNA evolved from an RNA base
What’s another example that DNA evolved from RNA?
Deoxyribose has to be made from ribose (RNA with ribose came first then DNA with deoxyribose is made from it)
DNA polymerisation
Extends DNA strand
Done at 3’ end of DNA
3’OH on the deoxyribose of DNA reacts with the the first phosphate of the dNTP (the phosphate attached to the base) releasing a pyrophosphate whilst phosphodiester bonds form ( connect the deoxyribose sugar with the phosphate) - see slide diagram
Catalysed by DNA polymerase
DNA polymerase
Can only use ssDNA as a template
Needs RNA primer - hybridises to ssDNA template and is used as starting point for polymerase
Another RNA world relic
The synthesis of DNA always starts with an RNA primer binding to the ssDNA so that the DNA polymerase can work
What does DNA polymerase do
DNA polymerase use dNTPs as building blocks & only synthesise from 5’ —> 3’ end
How do DNA polymerase a stay on the ssDNA template?
DNA polymerase stay on the template
They are processive
Stay on with a beta-clamp (protein complex that keeps polymerases on ssDNA template until the end)
DNA polymerase proofreading property
Polymerase recognises mismatch. Reverses, (newly synthesised strand peels off and sticks into expnuclease active site which hydrolyses phosphodiester bonds so nucleotides are released until mismatch is removed)
Then DNA polymerase moves forward again
Statistics with and without proofreading
Without - 1 error per 10^5 copied nucleotides
With - 1 error per 10^7 copied nucleotides
what is the process of mismatch repair?
See ppt slide
If there is a Mismatch, enzymes scanning DNA will recognise it
- DNA glycosliase removes base
- Endonuclease lyses phosphate backbone
- Repair DNA polymerase (different to one used in replication) fills the gap
- DNA Ligase repairs phosphodiester bond and makes continuous phosphate backbone
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Helicase unwinds DNA and separates DNA strands (1000bp/sec)
Polymerase works in 5’ —> 3’ direction, copying the leading strand
Copies the lagging strand in Okazaki fragments (still in 5’ —> 3’ direction)
Roughly How long are okazaki fragments?
1000 base pairs
Lagging strand replication
- RNA primer required to make a starting point for polymerase (primase associated with helicase and makes RNA primer 11 nucleotides - RNA world relic btw)
- Then DNA polymerase can extend the RNA primer and make ssDNA on top of the template strand until it hits the RNA primer used in the previous synthesis of the last Okazaki fragment
- RNase H degrades RNA primers
- a DNA polymerase (different to the DNA polymerase previously spoken about) extends Okazaki fragments
- Gap between Okazaki fragment’s where the RNA primer was is then filled in by DNA ligase (joins Okazaki fragments)
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Prokaryotic replication
Replication can be at any time during the life cycle of the cell
DNA is circular and has a single origin of replication
In bacteria we often see theta structures (see replication)
Eukaryotic replication
Replication always in S phase
Multiple origins of replication (
Linear DNA
What do eukaryotes have which is a major disadvantage in replication and why?
Linear chromosomes
Means there is an end replication problem - every time you copy a linear piece of DNA the copy of the lagging strand becomes a bit shorter
Why are the RNA primers removed?
Because they contain RNA nucleotides and DNA needs DNA nucleotides
What do all DNA polymerase enzymes do?
Add nucleotides to 3’ OH ends of DNA
Explain the end replication problem
RNA primers from left to right are in a 5’ —> 3’ direction. (The complementary DNA strand from left to right is in a 3’ to 5’ direction)
The DNA polymerase moves alone the strand with the RNA primers in it and adds a nucleotide to the 3’ end of the RNA primer. But once u get to the end of the lagging strand there is no 3’ end.
You cant just remove the parent. Strand overhang because that is still information needed
Solution to the end replication problem
Telomerase enzyme - has its own RNA template
Telomerase works on the parent strand - elongating the 3’ end.
The last two bases of the telomerase enzyme pair up with the last two bases of the parent strand and makes a newly synthesised DNA strand
Then the primase lays down a primer at the end so now there is a 3’ OH end.
RNAse H removes primer
Now we can remove parent overhang cos it was synthesised by telomerase (FEN1 cuts out overhang)
First step of solution to end rep problem
Telomerase extends parental strand using RNA template inside it (the telomerase)
Second step of solution to end rep problem
Telomerase makes telomere extension (RNA guided DNA synthesis)
Third step of solution to end rep problem
Primase makes RNA primer which attaches to the extended parent strand
Fourth step of solution to end rep problem
DNA polymerase extends RNA primer (5’ to 3’ direction) + ligase connects phosphate backbone
What’s an RNA world relic in the solution mechanism to the end replication problem?
The telomerase has its own RNA template which is used to help direct the extension of DNA
(Suggests RNA came before DNA)
What transcribes DNA into RNA?
RNA polymerase
What building blocks does RNA polymerase use?
NTPs
Nucleoside triphosphates
RNA polymerase properties
Only synthesises in 5’ to 3’ direction
Uses double stranded DNA as a template - uses Only one strand (template strand) is copied
Tightly regulated initiation of transcription (because when the initiation takes lace determines which genes turn on) - regulates complex initiation procedure
Start from initiation site doesn’t require primer
What does RNA polymerase do?
Extends the RNA at the 3’ OH of the ribose with an NTP
(Get phosphodiester bond) see ppt slide
Release of pyrophosphate
Transcription through electron microscopy
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Christmas tree like structures
Multiple polymerases move along a single gene
Initiation of transcription
Polymerases move along gene until they get to region where they stop translation
RNA produced gets longer and longer
So then can predict the top of the Christmas tree is an area where transcription is initiated (promoter)
And the bottom is where transcription is terminated (terminator)
How is transcription initiated in prokaryotes?
- DNA has two motifs upstream of the transcription initiation site
- Sigma factor (protein) scans DNA for the motifs, recognises them and binds to the major groove of the DNA
(Sigma factor recruits RNAP - RNA polymerase) - RNAP separates two strands - unwinds 17bp of DNA to form transcription bubble. (See ppt slide)
- In the loop you get synthesis of RNA. Once this is initiated the polymerase (RNAP) moves further down the DNA elongating it in a 5’ to 3’ direction, the sigma factor dissociates and is left behind.
= produces RNA
What are the two motifs upstream of the transcription initiation site in prokaryotes?
TTGACA
TATAAT
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Explain 6 steps of transcription initiation in eukaryotes
- DNA contains a TATA box (hognes box)
- TATA binding protein recognises TATA box.
- TATA binding proteins recruits TFIIA and TFIIB
- Then TFIIB recruits the RNA polymerase II (Pol-II). This polymerase comes with transcription factor two F (TFIIF)
- TFIIE then joins and this recruits TFIIH (this one is a helicase and a kinase). So when TFIIH is bound a separation of the strands occurs.
- Transcription bubble forms and Pol-II (the polymerase) is phosphorylated (by TFIIH and upon signals from upstream elements) and THIS signal causes transcription to be initiated
- Once phosphorylation occurs, Pol-II dissociâtes from TFIID complex and moves over the DNA to make a copy of the RNA (aka. Pol-II transcribes RNA from 5’ to 3’)
Name two things in eukaryotic transcription that contribute to informing what position transcription should start from
Regulatory elements upstream on the DNA
Enhancer sequences/regions
What is the Tata binding protein (TBP)
One of the 9 subunits that makes up the transcription factor IID complex (TFIID) (this complex of proteins binds to the TATA box)
What’s at the start of transcribed prokaryotic RNA?
5’ end of the transcribed RNA is a triphosphate and a purine base (guanine or adenine)
What’s at the start of transcribed eukaryotic RNA?
5’ cap
Triphosphate connected to a guanine which is methylated at position 7.
Also there are methylations of the 2’OH in the first one or two ribose sugars of the nucleotides in the RNA.
Importance of the 5’ cap?
Three things
Without it splicing cannot occur (introns cannot be removed)
Required for translation (without it the ribosome cannot bind to the transcript and scan for the first start codon)
Increases RNA stability
Termination in prokaryotes
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What’s at the beginning and end of the mRNA either side of the ORF in prokaryotes?
5’ UTR (untranslated region)
3’ UTR
In prokaryotic termination during transcription, where does the termination signal occur ?
In the 3’UTR of the mRNA
Explain Rho-independent termination in prokaryotic transcription
Specific inverted repeat in the 3’UTR and as soon as this region is transcribed it creates a hairpin.
The hairpin is recognised by protein NusA which binds to it and stops the polymerase from further transcription down the strand
Explain Rho-dependent termination in prokaryotic transcription
Rut (Rho-utilization site) sequence is recognised by protein Rho which binds to this part of the RNA
Rho moves upwards towards the polymerase to block transcription
Two mechanisms of termination in prokaryotic transcription
Rho independent termination
Rho dependent termination
Explain termination stage of eukaryotic transcription
Termination signal in 3’UTR = PolyA signal
Enzyme binds to the polyA signal, cleaves it and stops transcription
(Cleavage occurs at the end of the transcript after the ORF - downstream of the polyA signal (the AAUAAA box) which comes after the ORF)
Poly A polymerase adds 200-250 adenosines to the 3’ end of the RNA (PolyA tail)
What is the polyA signal?
AAUAAAA
How is a polyA tail made?
At the end of eukaryotic transcription, Poly A polymerase adds 200-250 adenosines to the 3’ end of the RNA
What does the polyA tail do?
Increases RNA stability
What is splicing
Removal of introns from preRNA
How often does splicing occur in prokaryotes and eukaryotes?
Frequently in eukaryotes
Occasionally in prokaryotes
2 steps to splicing
See ppt slide
Splicing
Catalyst?
What it involves?
Catalysed by spliceosome (a ribozyme) - RNA world relic
Involves 5 small nuclear RNAs (snoRNAs) and proteins
Self splicing introns
some introns which can splice themselves (RNAs which can splice themselves). Sometimes found in bacteria.
Prokaryotes vs eukaryotes
Spatial/temporal separation
Prokaryotes - transcription and translation can occur simultaneously
Eukaryotes - transcription only occurs inside nucleus and translation only occurs in cytosol (processes are spatially separated and therefore also temporally separated)
What does splicing require ?
Splicing only occurs on transcripts with a polyA tail and a 5’ cap
RNA world relics in DNA replication
Deoxyribose is made from ribose
dTDP is made from dUDP
DNA polymerase uses RNA primers
Telomerase uses an RNA template
RNA world relics in transcription
mRNA, termination hairpin 5’ cap PolyA Spliceosome (it’s a ribozyme) Self splicing introns
