RR12: Translation II

Question 1

what does the polyA tail allow for in mRNA?

Answer 1

• longer poly A tail= more stable mRNA
• nucleotides buy mRNA time as it gets degraded from the 3’ to 5’ direction
• The more polyA you have, the more protein you have as well (PABPC)
• PABPC also blocks the ability of those RNAases to chew the molecule up
• The polyA tail also allows for extra PABPC that can interact with the 5’ end (eIF4G)
• Contributes to a stabilisation of the mRNA but also increases efficiency of translation
• Longer poly A tail: translated much longer

Question 2

what is the scanning complex, what does it do and when does it stop?

Answer 2

• RNA helicase of eIF4A uses ATP and scans through the 5’ untranslated region (100 nucleotides), in doing so it unwinds structures that might’ve been there
• that scanning complex (not sure if eIF4E is there too) continues to process along the 5’ end until it reaches the start codon
• AUG recognized by tRNAi MET with anti codon interaction

Question 3

what happens once the pre initiation scanning complex recognises the start codon?

Answer 3

• That interaction with anticodon gives rise to major changes int eh complex
• GAP associated with the scanning complex in eIF5 that will act upon eIF2-GTP that is interacting with tRNAi MET, and will cause eIF2 to hydrolyse GTP to GDP
• Major conformational change, all of the subunits/factors leave the complex
• you end up with the small subunit with the tRNAi MET bound at the P site, associated with the mRNA over the AUG start codon
• The large subunit can come in and recognize that structure to give rise to the 80S complex, ready to fire

Question 4

what are tRNAs associated with?

Answer 4

• The tRNAs are in association with EF1 alpha-GTP (elongation factor 1 alpha, GTP binding protein)

Question 5

how do tRNAs get in contact with the A site?

Answer 5

they just diffuse

Question 6

what happens if the tRNA anticodon does not match the codon?

Answer 6

• if tRNA enters the A site associated with EF1 alpha-GTP and it cannot base pair correctly, it will diffuse out of that site and nothing will happen

Question 7

what happens when the tRNA anticodon matches with the codon?

Answer 7

if it works, there is a conformation change associated with the hydrolysis of GTP in eF1 alpha , and GTP becomes GDP
* Changes the conformation of the ribosome and the tRNAs and their proximity within the ribosome.
* the tRNA is shifted a little bit —> the AAs linked to those tRNAs come into close proximity, so close that they can interact

Question 8

what parts of the adjacent amino acids interact to form the new peptide bond?

Answer 8

• because of that change in the ribosome and the position of the two AAs bound to tRNAs, a catalysis can take place where the alpha amino group on the AA attached to the tRNA on the A site can interact with the terminal carboxyl group of the AA on the P site
• This bond links the AA attached to the tRNA on the A site to the AA attached to the tRNA at the P site (in this case the Methionine associated with tRNAi)

Question 9

what enzyme helps the formation of a new peptide bond?

Answer 9

peptidyltransferase

Question 10

what is significant about this reaction?

Answer 10

• formation of that bond takes place in the ribosome, through an RNA mediated catalysis
• If you examine structure of the ribosome where the actual peptidal transfer takes place, its in a region where very few proteins are present, but the rRNA is in that region
• Argues that this catalysis is not associated with any clear protein/enzyme catalytic site but rather with the presence of the large ribosomal RNA
• if we peel away the proteins and only leave the RNA, the catalysis of the peptide bond would still work
• rRNA folds up into secondary structures that can give rise to enzymatic activities by the way they fold around their targets
• Again suggests that earliest forms of life depended on RNA

Question 11

what is a ribozyme?

Answer 11

RNA with catalytic activity

Question 12

what does the GTP hydrolysis in EF1 alpha cause?

Answer 12

the conformation of the ribosome changes, which translocates along the mRNA the distance of one single codon (3 nucleotides)

Question 13

what mediates the translocation?

Answer 13

• That translocation event is tightly regulated and monitored by another EF called EF2-GTP

Question 14

what do GTP proteins ensure?

Answer 14

• Through hydrolysis, the system can’t go backwards
• GTP proteins ensure that the system goes forward and can’t slip and go back

Question 15

what is the rate of elongation?

Answer 15

3-5 amino acids per second

Question 16

when does the elongation stop?

Answer 16

when a stop codon is reached

Question 17

what can interact with the stop codons?

Answer 17

there is no tRNA that can interact with the stop codons
but there is a protein that looks like a tRNA that can fit into the A site and can interact with those stop codons, in the N terminus
* The protein fits in there and will give rise to chain termination
* Protein is called a release factor: eRF1 (eukaryotic release factor 1)

Question 18

what happens when eRF1 binds to the A site?

Answer 18

• eRF1 is associated with GTP binding protein called eRF3, and once eRF1 Interacts with stop codon, eRF3 will hydrolyse GTP to GDP, changes in the ribosome which ultimately ends up cleaving the polypeptide chain

Question 19

what happens after the polypeptide chain is cleaved in termination?

Answer 19

• on the chain is cleaved, more changes happen on the ribosome: the subunits dissociates, and again as soon as the small subunit leaves, initiation factors, 1, 3, and 1A will associate with the small subunit so that it can’t come back together with the big subunit
• Ready to participate in another reaction

Question 20

what do more ribosomes on an mRNA mean?

Answer 20

more ribosomes=more translation=more proteins

Question 21

how can the different complexes be visualised?

Answer 21

• we can separate the complexes by using ultracentrifugation or just centrifugation
• If you run cytoplasmic extract through gradient (sucrose) you can separate each of the complexes after centrifugation (long)
• Heavier complexes towards the bottom and the lighter ones at the top (density)
• If we take fractions starting from top to bottom we can see the graph
• Lighter: initiation factors, small ribosomal subunit, the 60S ribosome (the large subunit)
• You can also separate out the 80S initiation complex
• Lots of them: the limiting step
• mRNA that has lots of ribosome is very heavy, and it is being heavily translated
• polysomes /polyribosomes