Genome chemistry, epigenetic & DNA methylation (meg) Flashcards
What can genomes be made of?
DNA or RNA
Name 5 viruses made up of single stranded RNA
HIV
Flu
Ebola
Polio
Measles
Name 2 viruses that are made up of double stranded RNA
blue tongue
rotavirus
Name 3 viruses that are made up of single stranded DNA
M13
ɸX174
sea water viruses
Name 4 viruses that are made up of double stranded DNA
Complex viruses:
- Herpes
- Smallpox
- bacteriophage T4
- lambda phage
What are the genomes of cellular made of?
Double stranded DNA
What is the corona virus genome made of?
Single stranded RNA
What are the 2 levels of nucleic acid chemistry?
- Individual bases = nucleotides
- Higher order structure (double helix)
What is this picture showing?
RNA nucleotide (need to be able to draw)
What defines an RNA nucleotide?
the hydroxyl group at the 2’ position on the ribose sugar
How many carbons are there in ribose / deoxyribose sugars?
5
What is the difference between an deoxyribose sugars and ribose sugars?
Deoxyribose = oxygen is missing from 2’ prime position so just a single hydrogen = resulting in important consequences and making DNA much more stable than RNA
What makes an deoxyribose sugar more stable than a ribose sugar?
hydroxyl group on RNA can catalyse the hydrolysis of the phosphodiester bond
Name all the pyrimidine bases?
C
T
U
Outline the structure of pyrimidine bases
2 nitrogens and 4 carbons in pyrimidine labelled counter clockwise
Name the 2 pyrimidine bases in DNA
Cytosine
Thymine
Outline the structure of cytosine and thymine
Cytosine = carbonyl group (C=O) at position 2’, at 4’ position = amino group
thymine = same carbonyl group at 2 position + carbonyl group at 4’ position and at 5’ position = methyl group
In terms of bases, what is the difference between RNA and DNA?
In RNA, it is uracil instead of thymine (DNA)- both pair with adenine
Describe what was discovered (2023 nobel peace prize in medicine)
In RNA instead of uracil- many bases are modified to have pseudo-uracil
What is pseudo-uracil?
looks like uracil but instead of having base attached to ribose sugar through nitrogen, it is attached through carbon
How is the base attached to ribose sugar through carbon in pseudo-uracil?
Enzyme enables this- rotates the base and re-attaches through the carbon atom
Why was this discovery important?
- mRNA with pseudo uracil in it translated more efficiently than non-modified mRNA
- cells don’t promote an inflammatory response- way of discriminating between viral DNA which is unmodified and cellular DNA which is modified
Why did this discovery win the 2023 Nobel Peace Prize?
all RNA COVID vaccines used pseudo-uracil instead of uracil- it worked because without pseudo uracil much less protein would have been produced and the cells would have promoted an inflammatory response
Name the 2 main differences between RNA and DNA
- DNA has thymidine (thymine) - RNA has uridine (uracil)
- 2’ position of ribose sugar in DNA has a hydrogen atom while RNA has a hydroxyl group
Describe the structure of Deoxythymidine triphosphate
- 3 phosphate groups
- Thymidine- 5’ position has methyl group and carbonyl groups at 2’ and 4’ positions
- No hydroxyl group on sugar = DNA nucleotide
- Carbon atom present at 2’ position on deoxyribose sugar
What are the 3 key features of nucleic acids?
- Polarity = 5’ end distinct from 3’ end
- Bases are linked by phosphodiester bonds = 2 ester linkages
- Glycosidic bond- bond between 1’ carbon on ribose sugar and 1’ position nitrogen on base
What is the direction of a nucleic acid sequence?
5’ to 3’ direction
What is between bases in double stranded nucleic acids?
hydrogen bonds
How many bonds are between each type of base pair?
T + A = 2 hydrogen bonds
C + G = 3 hydrogen bonds
Describe the arrangement of double stranded DNA
2 DNA strands have an antiparallel arrangement (one strand 5’ to 3’ and other strand is 3’ to 5’) and are wrapped around each other in plectonemic coil
Why is DNA a good molecule to encode genetic information and make stable genomes?
- DNA is more stable than RNA because of the presence of 2’ OH group in ribose sugar in RNA makes RNA alkali labile (= it breaks up)- 2’ OH acts as a catalyst for hydrolysis
- Hydroxyl group in presence of alkali could cause nucleophilic attack e.g. on a phosphate and in doing so, it would displace an oxygen next to the phosphate group- could result in phosphodiester backbone being cleaved and in turn impact the condition of the oligonucleotide - DNA has thymine instead of uracil- cytidine is unstable and in the presence of thymine allows the products of cytidine instability to be identified and removed = repaired
Define: cytidine
= nucleotide containing cytosine
what is an oligonucleotide?
= short chain of nucleotides
what is cytosine deamination?
hydrolytic reaction = converts cytosine into uracil as amino acid is converted into a carbon r group
Is cytidine deamination a physiological process or a non-physiological process?
non-physiological process
What is the difference between physiological and non-physiological processes?
physiological = characteristic of or inherent to the normal functioning of living organisms
Explain how cytosine deamination is different in DNA and RNA
Difference lies in the outcomes of the process:
RNA = end up with uracil
In DNA- have thymine instead of uracil, but when cytosine deamination happens, we get uracil. The cell immediately knows that uracil should not be in DNA due to presence of thymine:
- The uracil is identified and repaired back into thymine
- So presence of thymine in DNA to enable cell to recognise the product (=uracil) of cytosine deamination
Name the enzyme that removes uracil in DNA and how does it do this?
uracil glycosylase- breaks glycosidic bond between sugar and the uracil = 1st step of base excision repair = BER
What does base excision repair (BER) also do?
Removes oxidised and alkylated bases
Describe the process of BER
BER removes the U and restores C:
1. By cutting the glycosidic bond = get apurinic/apyridinic site
- Enzymatic machinery comes along and removes apurinic/apyridinic site
- Repairs it and puts in a thymine- 2 types of repair reaction:
- Long patch
- Short patch
What came before the other- RNA or DNA?
Good evidence to suggest that RNA came before DNA
Outline the evidence to suggest that RNA came before DNA
- both can assemble on the basis of complementarity + store info but only RNA is significantly catalytically active
- all descended from RNA molecules- RNA performs both catalysis as starting point for life + in lab you can evolve RNA molecules = can make self-replicating RNA molecules
- DNA precursors are made from RNA precursors by ribonucleotide reductase
- 2 different thymidylate synthetases that methylate dUMP in many organisms suggesting T evolved twice
- PBS1 and PBS2 phage of B.subtilis contain U instead of T in their DNA- suggests its an ancestor for DNA
What does ribonucleotide reductase do?
Reduces ribonucleotides
Name some more reasons why double-stranded DNA is a good choice to encode genetic information
- Double stranded DNA does not fall apart when phosphodiester backbone nicked since it is stabilized by hydrogen bonds and by hydrophobic interactions between the faces of the individual base pairs
- Nucleotide bases are protected from aqueous phase by phosphodiester backbone- in DNA = potentially reactive groups attached to bases e.g. amino groups, carbonyl groups but in double helix these are protected by phosphodiester backbone unlike single-stranded nucleic acids
- Double helix can be melted under physiological conditions- if it didn’t melt then genetic info couldn’t be accessed and transcribed- perfect balance between stability and instability
Name the 3 different double helices that double stranded nucleic acids can form
A DNA
B DNA
Z DNA
What is the main form of double stranded DNA helix?
B-DNA
Describe the structure of B DNA and why is this beneficial?
Have a minor groove (can’t see the bases) and a major groove (can see bases)- good because transcription factors can read base sequence
Do this by binding through the major groove
How is Z DNA formed?
alternating purines and pyrimidines
What 2 types of interaction stablises the double helix?
Both non-covalent interactions:
1. Hydrogen bonds between bases
2. Hydrophobic stacking interaction between faces of the bases
Why are hydrophobic stacking interaction between faces of the bases stronger at high salt concentrations?
stronger at high salt concentration
if temperature is raised what will happen to double stranded DNA?
Double stranded DNA will melt into 2 strands if temp is raised
After melting double stranded DNA can this be reversed?
reaction reversible = hybridisation = 2 double strands will bond back together
Why is nucleic acid hybridisation fundamental in biology?
important in mRNA translation, tRNA transcription- codon (mRNA) and anticodon (tRNA) interactions, homologous recombination = hybridisation of single strands of DNA
Why is nucleic acid hybridisation fundamental in molecular genetics?
- PCR- depends upon hybridisation of the primers,
- Southern blotting- depends upon hybridisation,
- FISH- depends upon hybridisation of single stranded probe with denatured single stranded chromosomes,
- DNA sequencing- primers are annealed to single stranded DNA (same as PCR)
What is this picture showing?
Absorption at 260nm increases as double stranded DNA melts = hyperchromicity
What is DNA melting temperature sensitive to?
GC content
Why is melting temperture sensitive to GC content?
GC rich DNA will melt much more at a higher temperature than AT rich DNA- because there are more hydrogen bonds between guanine and cytosine base pairs
is cytidine deamination mutagenic?
Yes but is sometimes exploited biologically in physiological processes i.e Cytidine deamination causes mutations but the consequences of mutational processes are part of normal physiology of organisms
What group of enzymes catalyse cytidine deamination?
cytidine deaminases
Explain what cytidine deaminases are involved in
- RNA editing- important in birds also
- Innate immunity to retroviruses and transposable elements- so our cells have systems for protecting themselves
- Adaptive immunity = production of antibodies and antigen specific T cells- cytidine deaminases are used by adaptive immune system as part of normal process immunoglobulin gene diversification (=antibody diversification)
- Cancer- cytidine deaminases sometimes get mis expressed = mutations that give rise to cancer
How were cytidine deaminases discovered?
in RNA editing
Name the 2 forms of the apolipoprotein B (ApoB)
ApoB100
ApoB48
Where is ApoB100 synthesised?
Liver
Name the 2 domains on ApoB100
lipoprotein assembly
LDL receptor
What is the ApoB100 responsible for?
transporting lipids from liver to muscle tissues for production of energy
How do ApoB100 carry out their function?
LDL receptor domain on ApoB100 is bound to LDL receptor on tissue (particularly on muscle)- it enables muscle to take up the lipid on VLDL synthesised by the liver
Where is ApoB48 synthesised?
Small intestine
does ApoB48 have LDL receptor domains?
48% of protein lacked LDL receptor domain
Name the domains in ApoB48
lipoprotein assembly domain only
What is the ApoB48 responsible for?
carrying lipids from the gut back to liver
required for synthesis of chylomicrons by gut
Define LDL and VDL
LDL = low density lipoprotein
VLDL = very low density lipoprotein
What was the explanation for the 2 forms of ApoB?
The full length message encoded the Apo-B100 with the LDL receptor binding domain- it included all of the sequence corresponding to the exons in the gene
Then mRNA encoding the Apo-B48- had same length but one residue of cytidine in mRNA sequence which had been deaminated to uracil
- Result of deamination + presence of uracil = CAA codon (= glutamine) was converted into a stop codon by APOBEC-1- so abbreviated that position giving rise to Apo-B48
What is AID?
= activation induced deaminases (protein)
What are AID important in?
immunoglobulin gene hypermutation
Describe immunoglobulin gene hypermutation
1st produce antibodies they have low affinity but in mature B cells IG hypermutation takes place and the genes encoding the antibodies get further mutated- as a result of this = antibody response matures and become progressively of higher affinity