B: Master document Flashcards
What is the difference between a purine and pyrimidine?
A pyrimidine is a heterocylic aromatic ring like benzene which a nitrogen atom in the first and third carbon position.
Purine = pyramidine with imadazole ring fused to it.
Difference between protein coding gene and protein non-coding genes?
Non-coding genes are transcribed but do not undergo translation - becoming rRNA, tRNA, microRNA…
Protein coding genes are transcribed then spliced to create mRNA purely composed of protein encoding genes - then moves out nucleus to cytosol where it undergoes translation in ribosomes.
How do ribose and deoxyribose differ?
Ribose has OH attached to 2nd and 3rd carbons. Deoxyribose does not have OH attached to 2nd carbon, but has an H group.
Precursors of DNA and RNA
RNA = ribonucleotide 5’ triphosphates ATP, CTP, GTP, UTP.
DNA = deoxyribonucleotide 5’ triphosphates dATP, dCTP, dTTP, dUTP
What is RNA secondary structure and how does it happen?
When there are complementary bases within single stranded RNA and they pair with each other - intra strand base pairing.
E.g. stem loops, pseudo knots, hairpins.
key aspects of DNA structure
1- hydrogen bonds
2- antiparallel
3- right handed helix
4- major groove and minor groove
5- roughly 10.5 bp per rotation
6- Double stranded, double helix
7- bases project into the middle
8- phosphate backbone
Differences between DNA and RNA
Precursurs - uracil and thymine
Ribose and deoxyribose
Single + double stranded
RNA - intra base pairing
RNA - mRNA, rRNA, tRNA
DNA - genes
How can DNA be denatured
- high pH, 0.1N NaOH
- Heat
- Enzymes
DNA melting temperature
- Breaking double bond between AT - 2 degrees
Breaking triple bond between CG - 4 degrees
Almost all DNA strands are separated at 95 degrees - become single stranded.
DNA annealing definition
When two single strands of complementary DNA are free in a solution, strands will bind to each other in their complementary areas as long as conditions are suitable.
Explain how RNA polymerase synthesises RNA
- RNA polymerase starts at promotor site and transcription starts at +1 site.
- RNA polymerase either binds directly or indirectly to the promotor.
- RNA polymerase recruits RNA precursors - NTPs.
- RNA polymerase can synthesise RNA by using DNA as template
- RNA polymerase synthesises RNA from 5’ to 3’ direction.
- RNA polymerase catalyses the formation of 5’-3’ phosphodiester bonds between phosphate on 5’ carbon on NTP and 3’ OH on another nucleotide and released pyrophosphate.
Function of DNA topoisomerase?
Relieves supercoiling of DNA in front of RNA polymerase, and restores proper level of coiling to DNA behind RNA polymerase.
Explain how gene is transcribed into mRNA
- RNA polymerase initiates transcription by producing RNA
- Introns are removed by RNA splicing, and exons are rejoined in various ways to make different versions of mRNA to code for different versions of the protein.
- Strand is cleaved at polyA adenylation site on 3’ end (AAUAAA)
- Enzyme PolyA polymerase adds hundreds of adenines (polyA tail)
- 7 methyl guanine is added to 5’ end via 5’ to 5’ triphosphate bond –> mRNA is capped
What is the +1 site
Position on DNA where RNA polymerase begins to synthesise RNA
Terminator site in prokaryotes
Prokaryotes RNA forms a specific shape causing the RNA polymerase to dissociate from the DNA
Transcription bubble
When RNA polymerase binds to promotor, DNA melts forming a transcription bubble. 14bp wide.
Function of CAP
Translation initiation, protection, transport, splicing
Function of polyA tail
Transport, protection + termination.
Difference between mRNA in euk and prok?
Euk - splicing, cap, pokyA, not coupled.
Prok - Ribosome binding site, coupled.
Describe the structure of the nucleosome
Histone octamer + DNA surrounding it.
Describe the structure of histones
Histones are proteins that DNA is wound around.
Histone octamers are H3, H4, H2A, H2B.
H3 forms dimer with H4
H2A forms dimer with H2B.
H1 is not part of nucleosome or histone octamer, it is a linker protein that condenses nucleosomes and draws them together.
DNA synthesis
- DNA polymerase uses one DNA strand as template to synthesise complementary strand.
- DNA polymerase cannot synthesise DNA from scratch - can only synthesise DNA onto 3’ end of an existing pieces of DNA or RNA called a primer.
- DNA polymerase can only attach an incoming dNTP onto 3’OH group of deoxyribose, thus DNA polymerase synthesises DNA in 5’-3’ direction.
- DNA polymerase also catalyses formation of phosphodiester bond between phosphate on 5’ carbon of incoming dNTP and 3’OH on primer, generating 5’-3’ phosphodiester bond.
- High energy dNTP is consumed, pyrophosphate is released.
Proteins that work to repair mismatches
- MLH1, MSH2, MSH3, MSH6, PSM1, PMS2.
These proteins excise mismatch and re synthesise DNA.
Initiation of DNA synthesis
- Specific proteins bind to origin or replication. In eukaryotes, protein complex called ORC (origin recognition complex) binds to ori.
- Proteins at ori recruit additional proteins and melt DNA at ori.
- Helicases unwind DNA and form a replication bubble at ori. Single stranded binding protein binds to single stranded DNA and keeps it single stranded to enzymes can come in and work on it to replicate it.
- Proteins at ori recruit proteins that will replicate DNA
- RNA primer is synthesised at ori by DNA primase. When DNA is opened up, supercoiling builds up in areas flanking the opening. These needs to be relieved by Topoisomerase I and II (DNA gyrase in prokaryotes)
- After primer is synthesises, DNA polymerase can start DNA synthesis in 5’-3’
direction.
