topic 1.2 replication + transcription + translation Flashcards
1
Q
explain DNA replication
A
- initiator protein recognises DNA sequence where synthesis begins
- topoisomerase starts DNA unwinding, binding ahead of the replication fork to relieve the strain placed on the unravelling double helix
- helicase unwinds DNA
- single-stranded binding proteins bind to single-stranded DNA so they remain separated, prevents them from degrading
- primase attaches a small RNA primer to the single-stranded DNA
- DNA polymerase makes new strands by reading the template and adding new complementary bases
- after synthesis is finished, RNase digests the old RNA primer
- RNA primer is replaced by nucleotides
- Ligase catalyses the formation of phosphodiester bonds between okazaki fragments (lagging strand)
- This is happening simultaneously with the other strand so what results is 2 identical double-stranded DNA.
2
Q
how do you tell the lagging strand and leading strand apart?
A
- leading strand synthesises in the same direction as the replication fork, in one piece NOT FRAGMENTS
- lagging strand synthesises in the opposite direction to replication fork, in fragments (Okazaki fragments which must be ligated later)
3
Q
why is a primer needed for DNA pol to begin synthesising?
A
- DNA pol cannot begin synthesising from just the single-strand template.
4
Q
In RNA sequences, what is different about the nucleotide bases?
A
Instead of T, it is U
5
Q
differences between transcription and replication
A
- RNA pol instead of DNA pol
- only 1 of 2 strands acts as a template
- Template/antisense strand = RNA pol builds RNA complementary to this
- Coding/sense strand = RNA is identical to this (except T becomes U)
- Newly synthesised RNA transcript is displaced (cleaved) immediately
- RNA transcript is released from the DNA template as a single strand
- Each RNA transcript is only a short region of DNA (from start codon to end codon)
6
Q
describe transcription initiation
A
- Transcription factors help to position RNA pol at the promoter
- RNA pol slides along a DNA template, stopping when it recognises a promoter
- Transcription factors bind to the promotor, forming a pre-initiation complex (PIC)
- PIC includes/attracts RNA pol
7
Q
characteristics of promoters
A
- TATA box (TATAAA or variant, around 25 bp upstream of the start site, regulates transcription & binding site for TATA binding proteins/transcription factors)
- GC box (GGGCGG or variant, found in housekeeping genes e.g. histones/ribosomal proteins, 95 and 50 bp upstream respectively, binds specificity factor 1/Sp1)
- CAAT box (CCAAT or variant, around 80 bp upstream of the transcription start site, signals binding site for transcription factors)
8
Q
describe transcription elongation
A
- Transcription factors help to pull apart the DNA double helix and release RNA pol from the promoter when elongation begins
- RNA pol helps to unzip DNA during elongation process = transcription bubble, antisense/template strand, sense/coding strand
- RNA pol slides along the DNA template, with the transcription bubble moving along
- Elongation factors help to prevent the RNA pol from dissociating before it reaches the termination site of the gene.
- Enzymes (topoisomerase) help to remove superhelical tension that results from the RNA pol moving along the DNA double helix
- RNA pol adds complementary RNA nucleotides for each nucleotide on the antisense strand (U instead of T)
- RNA pol does not need primer to start synthesising the transcript.
9
Q
3 types of RNA pol (eukaryotes)
A
- RNA pol I (ribosomal RNA (rRNA))
- RNA pol II (all protein-encoding genes)
- RNA pol III (transfer RNA (tRNA), rRNA)
10
Q
describe transcription termination for RNA pol 1
A
Polymerase-specific termination factor binds to DNA downstream of the transcription complex.
11
Q
describe transcription termination for RNA pol II
A
- RNA pol II terminates around 0.5-2kb downstream of the end of the gene (after the gene ends) BUT mRNA transcript is already cleaved at the internal site at the actual end of the gene (before the termination of RNA pol II)
- Cleavage site occurs between AAUAAA (polyadenylation) sequence and a downstream GU-rich sequence (about 10-30 nucleotides apart)
- the AAUAAA sequence becomes the 3’ end of the transcript
- after cleaving, the 5’ exonuclease digests the excess overhanging strand that RNA pol II is still transcribing (as the termination site is downstream of the cleavage site)
- eventually, 5’ exonuclease catches up with the RNA pol II and RNA pol II detaches from the DNA template.
- transcription is terminated .
- at this point, the pre-mRNA must still undergo some modifications (see later card)
12
Q
describe transcription termination for RNA pol III
A
A series of U ribonucleotides on the RNA transcript triggers RNA pol III to release the RNA transcript and dissociate from the DNA template.
13
Q
What are the kind of post transcriptional modifications of mRNA in eukaryotes
A
- addition of 5’ 7-methylguanosine cap
- addition of 3’ polyA tail
- splicing out of introns
13
Q
Addition of 5’ 7-methylguanosine cap
A
- happens as soon as the mRNA transcript begins to synthesise/when transcription is still in progress
- 5’ cap is added to the first nucleotide
- 7-methylguanosine is a modified (methylated) guanine nucleotide
- Cap protects the transcript from being broken down when it moves to the cytosol for translation
- Cap helps the ribosomes to attach to the mRNA during initiation of translation
14
Q
Addition of 3’ polyA tail
A
- added at the same time as/after cleavage
- Cleavage exposes the 3’ end (AAUAAA/polyadenylated end)
- an enzyme adds 100-200 adenine (A) nucleotides at the 3’ end to form the polyA tail
- prevents the transcript from being degraded in the nucleus before it reaches the cytosol for translation
