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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