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Translation mechanism Flashcards

1
Q

What is initiation?

A

Binding of initiation factors to the small subunit (30S E.coli, 40S in eukaryotes). Find the AUG and plant a tRNA-Met.

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2
Q

What is elongation?

A

Factors bound to tRNAs deliver the next tRNA to the active site. tRNA recognizes codon. Shifting of the mRNA and amino acid joining.

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3
Q

What is termination?

A

Stop codon identified and release factors come in. Subunits are dissociated and mRNA is released.

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4
Q

What is recycling?

A

Dissociation of subunits and reuse for the next round of translation .

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5
Q

Is ribosome structure conserved?

A

Yes; prokaryotes and eukaryotes share a homologous core of RNA and proteins, although each also have different accessory proteins and rRNA extensions (rRNA much longer in eukaryotes).

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6
Q

Is ribosome sequence conserved?

A

No - different sequences in different species form very similar ribosome strucutres.

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7
Q

What is the role of the 30S / 40S subunit?

A

Decoding the mRNA (‘decoding centre’) and ensuring correct codon/anti-codon matching. Interacts with the mRNA and aminoacylated tRNAs.

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8
Q

What is the role of the 50S / 60S subunit?

A

Synthesis of the polypeptide strand - peptidyl-transferase domain (active site). Interacts with the aminoacylated tRNAs.

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9
Q

What is thought to be the function of the extra rRNA sequences?

A

Regulate the translation or export of the ribosomes via RBPs that bind them.

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10
Q

Which part of the ribosome is responsible for protein synthesis catalysis?

A

The rRNA - it is a ‘ribozyme’.

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11
Q

What is the CCA in a tRNA?

A

A 3’ sequence where amino acids attach.

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12
Q

What is the D loop in a tRNA?

A

A recognition site for aminoacyl synthetases (involved in aminoacylation of tRNA).

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13
Q

What is the T loop in a tRNA?

A

A recognition site for the ribosome.

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14
Q

What is the anticodon loop in a tRNA?

A

An RNA sequence that base pairs with the mRNA codon sequence. Also recognised by aminoacyl synthetases.

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15
Q

How long are tRNAs?

A

70-80 nts.

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16
Q

Why do multiple tRNAs recognise the same amino acids?

A

Because the genetic code is redundant.

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17
Q

How many tRNA synthetases are there?

A

22 - one for each amino acid.

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18
Q

What is the first energy dependent step of attaching amino acids to tRNA?

A

Amino acid + ATP binds to aminoacyl tRNA synthetase active site. ATP is hydrolysed to AMP + PPI (PPI released). AMP-amino acid intermediate is formed.

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19
Q

What is the second energy dependent step of attaching amino acids to tRNA?

A

‘Empty’ tRNA binds to the active site, releasing AMP and attaching to amino acid. Aminoacylated tRNA is released from the enzyme.

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20
Q

What are the 2 extra amino acids?

A
  • Selenocysteine
  • Pyrrolysine
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21
Q

How many tRNA binding sites are in a ribosome?

A

3: A, P and E.

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22
Q

What happens at the ribosome A site?

A

Aminoacylated tRNAs are loaded. Only remain there when there is stable base pairing between codon and anticodon.

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23
Q

What happens at the ribosome P site?

A

The amino acid is attached to the growing polypeptide chain (peptidyl transferase reaction).

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24
Q

What happens at the ribosome E site?

A

tRNA moves here when it loses its amino acid (translocation). It is then ejected from the ribosome.

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25
Where is the peptidyl transferase centre (PTC) in the ribosome?
Near the P site in the large subunit.
26
What does the large subunit L1 stalk do?
Binds tRNA in the E site; this binding is required for tRNA ejection. It is a flexible domain.
27
What does the large subunit central protuberance do?
Binds tRNAs in the P site. It is 5S rRNA and ribosomal proteins.
28
What does the large subunit L7/L12 stalk do?
Recruits translation factors (mostly GTPases). 'GTPase nucleation centre'. It is a flexible domain.
29
How does a ribosome know which AUG to start translation at in prokaryotes?
4 nt Shine-Dalgarno sequence (purine rich e.g. AGGA) 6-7 nts upstream of the AUG. This base pairs with the ribosome's 5' 16S rRNA (component of small subunit), anchoring it in the right place.
30
How does a ribosome know which AUG to start translation at in eukaryotes?
The first AUG downstream of the cap (ribosome scans for this). A Kozak sequence (CG rich) surrounding the start codon helps optimise translation by allowing effective ribosome binding (but not an initial binding site).
31
What is the 30S translation initiation complex?
- 30S ribosomal subunit - IF1/2/3 - GTP
32
What do prokaryotic initiation factors (IFs) do?
Help position the components of the initiation complex.
33
What is the function of IF2?
It is a GTPase and helps drive prokaryotic translation initiation forward. Also recruits tRNAfMet to the P site of the small subunit. (Forms GTP-IF2-tRNAfMet).
34
What happens once the 30S translation initiation complex has been formed?
It will bind to mRNA.
35
What is the function of IF1?
Binds to small subunit A site and blocks binding of tRNAs.
36
What is the function of IF3?
Binds to small subunit, mediating ribosome-mRNA interaction. Prevents binding of large subunit to small subunit.
37
What happens once the 30S translation initiation complex has bound mRNA?
IF3 is released, IF2 hydrolyses GTP, remaining IFs are released. Large subunit joins to form 70S initiation complex. Translation starts.
38
Why is the start methionine formylated?
- The amino group is blocked so it is not able to be incorporated into a growing polypeptide chain. Therefore it is only able to be the N-terminal amino acid. - The methionine still resembles a peptide, so the P site doesn't have to change.
39
What happens to the N-terminal formyl group once the polypeptide has been synthesised?
It is removed by a deformylase. This is because formylated polypeptides are less stable (could be a degradation signal).
40
What is the 43 pre-initiation complex?
- 40S ribosomal subunit - eIF 1/2/3 - initiator tRNA-Met (not formylated)
41
What is the function of eIF2?
It is a GTPase that binds tRNA-Met to the P site of the small subunit. (Forms GTP-eIF2-tRNAMet).
42
What is the function of eIF3?
Binds eIF4G in the cap-binding complex (a.k.a. eIF4F), mediating ribosome-mRNA interaction.
43
What is 'scanning'?
Once mRNA is circularised, hydrolysis of ATP stimulates the 43S pre-initiation complex to translocate in a 5' to 3' direction until it finds the start codon.
44
What is the function of eIF1 and 1A?
Assist in the scanning process.
45
What happens when the 43S pre-initiation complex stably binds the start codon?
- The 48S initiation complex forms. - The eIFs are removed via GTP hydrolysis by eIF2 (and eIF5B). Triggered by AUG recognition and large subunit joining. - The large subunit can then join this and the ribosome is assembled.
46
What is the function of the 5' cap?
- Prevention of degradation by 5' to 3' nucleases. - Promotion of translation by binding 43S pre-initiation complex.
47
What is the function of the polyA tail?
- Prevention of degradation by 3' to 5' nucleases. - Allow circularisation of mRNA and more efficient translation.
48
When is elongation initiated?
When the tRNA in the A site has a cognate interaction (acceptable) with the codon. The tRNAs fly in and out until this point.
49
How does the ribosome distinguish between a cognate and a near cognate codon-anticodon interaction?
There is an induced fit (specific nts flipped out) with a cognate pairing. (The difference between base pairing energies is not enough).
50
What is a cognate codon-anticodon interaction?
The first 2 bases of an anticodon are perfectly complementary (Watson-Crick base pairing), but a wobble base-pairing interaction (different base) is allowed in the 3rd base.
51
How does the ribosome distinguish between Watson-Crick and wobble base-pairing interactions?
(At the minor groove) Watson-Crick interactions (A-T and G-C) are similar in shape and surface properties. This shape is monitored by 3 highly conserved nts in the 16S rRNA.
52
What happens when a cognate interaction is established?
The ribosomal nts are 'flipped out' and insert themselves into the minor groove of the RNA helix (an 'A minor interaction'). They interact with the base pairings in a stable way; the closed conformation of the ribosome is stabilised. This interaction is energy costly, and can only be 'paid for' by a cognate match. The flipping out locks the tRNA into place.
53
How does paromomycin lead to misincorporation of amino acids?
It provides energy to stabilise flipping out of the ribosomal nts / promotes flipping out, even when Watson-Crick base pairing is not present (there is not a cognate interaction). The cell produces rubbish proteins that can lead to its death.
54
What is the function of EF-Tu (prokaryotes) / eEF1 (eukaryotes) GTPase?
GTP bound form binds 5' and 3' ends of tRNA and guides it to the A site of the ribosome via interaction with the L7/L12 stalk. A cognate interaction stimulates GTP hydrolysis, so the tRNA is stabilised in the A site and the EF-Tu is released. Only after this release can the peptidyltransferase reaction happen.
55
What is the function of EF-Ts (prokaryotes) / eEF2 (eukaryotes) GEF/GNRP?
'Recharge' EF-Tu. Binds EF-Tu and displaces GDP. Then EF-Ts is displaced by GTP.
56
How abundant is EF-Tu in E. coli?
VERY. Almost every tRNA is bound by EF-Tu, bar tRNAfMet.
57
Why is EF-Tu GTP hydrolysis slow?
It allows the system to check if the tRNA codon pairing is correct.
58
What is the peptidyl transferase reaction catalysed by?
The 23S rRNA! (Large subunit).
59
How does the amide bond form?
Nucleophilic attack of new (tRNA bound) aa's amino N (in A site) on polypeptide's carbonyl C (in P site). Polypeptide is transferred from P site tRNA to A site tRNA and then the whole thing must be shifted along 3 nts (translocation).
60
What is translocation?
Transfer of the A site tRNA / polypeptide to the P site. Allows reading of the next codon. Happens through shifting of mRNA within the small subunit.
61
How does translocation occur?
The P site tRNA that has been released moves to the E site spontaneously (higher affinity). Then the A site tRNA with the polypeptide moves to the P site spontaneously (higher affinity). Movement called a 'ratchet', involves 6 degree rotation of the ribosome small subunit. Ratcheting is spontaneous but reversible.
62
How is reversal of translocation prevented?
EF-G locks the subunits in the ratcheted state via GTP hydrolysis, so translocation is driven forward.
63
How does EF-G prevent the aminoacyl tRNA from moving back into the A site?
It has evolved to be a tRNA mimic - it occupies the A site itself because it has a similar shape to tRNA.
64
How do the antibiotics fusidic acid and viomycin inhibit translation?
The block (different stages of) EF-G activity.
65
Which stage of translation do antibiotics tend to target?
All of them! There are so many.
66
How does the antibiotic chloramphenicol inhibit translation?
Binds to the peptidyl transferase cavity and prevents binding of tRNA to the A site. Therefore nascent peptide cannot be transferred from the P site to the A site.
67
What happens in the prokaryotic ribosome exit tunnel?
It is narrow so the polypeptide folding is constrained, but once it binds to chaperone proteins in the solvent it can fold.
68
What happens in the eukaryotic ribosome exit tunnel?
Some chaperones can access the polypeptide within the tunnel via ribosome interactions to stimulate cotranslational folding and prevent unwanted aggregate structure.
69
How do macrolide antibiotics inhibit translation?
They block the exit tunnel by interacting with rRNA nucleotides in it, inadvertently blocking the peptidyl transferase reaction. Doesn't impact eukaryotic translation as their exit tunnel is narrower so the antibiotics can't get in.
70
What happens when the ribosome encounters a stop codon?
Release factors bind the stop codon in the A site (not tRNAs), and stimulate cleavage (hydrolysis) of the nascent peptide from the tRNA in the P site.
71
What is the eukaryotic release factor?
eRF1
72
What is the eukaryotic EF-Tu release factor equivalent (binds A site)?
eRF3
73
What are the prokaryotic release factors?
RF1 and RF2
74
What is the prokaryotic EF-Tu release factor equivalent (binds A site)?
RF3
75
What happens in termination after the nascent polypeptide has been cleaved?
GDP-(e)RF3 is recruited to the A site, which induces release of the (e)RF. Exchange of GDP for GTP and subsequent GTP hydrolysis is thought to release the (e)RF3. The ribosome is then disassembled from the mRNA by ribosomal release factor (RRF) and EF-G; the ribosomal subunits can then be reused.
76
How is the empty tRNA in the P site removed from the 50S subunit after translation termination?
Using (e)IF3.
77
Which molecule do release factors show mimicry to?
tRNA! (and EF-G). They all occupy the ribosome A site.
78
Which motif in the release factor promotes peptide release?
GGQ - same place as CCA.
79
Which motif in the release factor interacts with the stop codon?
PVT - same place as anticodon.
80
How is the peptide cleaved from the P site tRNA (amide hydrolysis)?
Water is allowed into the P site; RFs make a catalytic pocket with space for water. The active site usually has no space for water to enter and hydrolyse! Otherwise this would happen spontaneously all the time.
81
What type of bond links the tRNA and amino acid?
Ester bond.
82
Which bond is hydrolysed for nascent peptide release?
The ester bond between the tRNA and terminal amino acid.
83
What happens when a non-cognate interaction is detected?
The flipped out nts do not stable interact with the mRNA/tRNA, and the ribosome returns to the open conformation where there nts are stacked.

Gene Expression (9 decks)

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