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Gene to function - Eukaryotes > Lecture 23 - Eukaryotic Translation > Flashcards

Lecture 23 - Eukaryotic Translation Flashcards

1
Q

Very briefly define translation

A

Synthesis of polypeptides from an mRNA template

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2
Q

What is the RNA type that is also known as ‘the translator’

A

tRNA

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3
Q

What is the structure of the tRNA and how do it structures work with mRNA?

A 
Acceptor stem (Out the top) - where the correct peptide is bound
D loop and TYCG loop (y should be a potential sign there) - just adda  secondary structure to it, not important
Anticodon loop - where base pairing occurs with the cognate codon on the mRNA
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4
Q

Does tRNA contain many ‘unusual’ nucleotides? If so what do these reflect?

A

Yes

The post translation modifications that have occured

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5
Q

What is the name of the enzyme that attached tRNA to the corresponding amino acid

A

aminoacyl-tRNA synthetase

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6
Q

How does aatRNAsynthetase link the tRNA and amino acid?

A

Via the hydrolysis of ATP - this generates a phosphodiester bond to create the complex.

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7
Q

How often does an error occur in matching of amino acid to tRNA and why is this?

A

1 in every 10^4-5 couplings

Because the enzyme has a proof reading function so is very accurate

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8
Q

What else does aatRNAsynth do?

A

delivers the aminoacyl - tRNA complexes to the ribosome

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9
Q

Where are the ribsomes found?

A

Cytoplasm or on endoplasmic reticulum

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10
Q

What are some functions of the ribosome?

A
  1. Recognise the mRNA sequence with the codon complimentary to that of the anti-codon on the aa-tRNA
  2. (Specifically those on the ER) synthesis secreted and membrane bound proteins
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11
Q

Describe the overall structure of a ribosome, including what their made of

A

Large subunit and small subunit (60s and 40s)

Made of 60% RNA and 40% protein (mosyt abundant rna-protein complex in cell)

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12
Q

How fast do ribsmes elongate polypeptides?

A

3-5aa per sec

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13
Q

What do mutations in ribosomal components lead to?

A

Very specific symptoms, even though its a defect in something so essential to functionality

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14
Q

In humans, how many types of ribosomal RNAs are there? And ho wmany ribosomal proteins?

A

4 and 80

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15
Q

What are the 3 sites within the ribosome that the aa-tRNA passes through

A

a (aminoacyl site)
p (polypeptide site)
e (exit site)

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16
Q

What is the enzymatic ribosmal core called and what does it catalyse

A

Peptidyl transferase - catalyses peptide bond formation and therefore peptide transfer

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17
Q

Describe the process of the aaTRNA moving through the 3 ribosomal sites

A

aatRNA enters at A site
The amno group of it is positioned to attack the ester lonkage between the peptide chain and tRNA in the P site - this reaction is catalyed by the peptidyl transferase centre
The tRNA then receives teh peptide chain (while it’s still in the A site)
The new tRNA complex translocates to the p site
uncharged tRNA (without peptide I think - because A has stolen it from P site), can then move to E site and is released

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18
Q

What is the most highly regulated stage in euk translation?

A

Initiation

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19
Q

What are the translation proteins invovled in initiation?

A

Eukaryotic Initiation Factors

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20
Q

How many eifs are involved in initiation?

A

12 - and most have no bacterial homologue

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21
Q

Describe what is happening regarding the ribosomal subunits before initiation

A

They are kept apart by eif3 (bound to 40s) and eif6 (bound to 60s)
eIF3 is key for mRNA recruitment to ribo

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22
Q

What is the very first step in initation (generally, not the associated factors)

A

Positioning of 40s and 60s subunits, then recruitment of initiator methionyl tRNA to position at the start codon, AUG, of the mRNA

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23
Q

Describe the process of the addition of methionyl tRNA to 1 ribosomal subunit

A

40S is bound by eIF3 and eIfIA
This complex recruits a ternary complex (eiF2.GTP.met-tRNA^met
All together this forms the PRE INITIATION COMPLEX (called 43s PIC)
The methionyl tRNA is included in the ternary complex so is delivered by eIF2
60s is not involved yet

24
Q

What are the features of eIF2 that allow if to perform it’s function (and what is it’s function)

A

Has 3 subunits (alpha, beta, gamma)
It recognises uniquestructural features throughout the tRNA^met so it can recognise it an deliver it to the ribosome
The gamma subunit has GTPase activity

25
Q

How is mRNA recruited to the 43s PIC (general)

A

By the eIF4 cap binding complex

  • made of several factors
  • it makes multiple redundant contacts with the mRNA to reduce the danger of system failure if any single interaction disengages
26
Q

What is the mRNA cap?

A

it’s an inverted 7-methylguanosine group at the 5’ end of euk mRNA

27
Q

What makes up the eIF4 cap binding complex and what does each component do

A

eIF4E - bind the cap. HIghly regulated, but found to be de-regulated in many cancers
eIFG - serves as a scaffold (bind eIFE and eiF4A and mRNA)
eIF4A - a helicase that unwinds 2ndry structures in the 5’ untranslated region of the mRNA, to allow access by the 43s PIC
eiF4B - aids A. Interacts with eIF3 in the 43s PIC to bring the complex together.

28
Q

In eul mRNA normally monosystronic or polysystronic

A

mono - encodes single polypeptide.

29
Q

What is the genral term for what happens after the 43s pIC has bound to the cap?

A

mRNA scanning

30
Q

Describe the process of mRNA scanning

A

43s scans along mRNA until it find start codon - AUG

It may scan through suboptimal AUGs

31
Q

What makes a start codon strong/desirable/optimal

A

Optimal bases will flank it that conform to the ‘kozak sequence’

32
Q

What is the process called where the PIC leaves out suboptimal start codons in favour of better ones?

A

leaky scanning

33
Q

Is it possible to have cap independant lation?

A

Yes

34
Q

What must an mRNA have to be translated without a cap?

A

An IRES - a region with a complex secondary structure, not just a sequence.
It is recognised by proteins that allow recruitment of PIC without the cap.

35
Q

Describe the process that occurs after start codon recognition?

A

eiF2 hydrolyses GTP - therefore recognition process is now irreversible
eIF3 and 4 dissociate and scanning stops
What is left of PIC (eiFIA and eif2.GDP) is now sat over the AUG
The p site of the ribosome is now occupied by the met-tRNA^met
Now the large subunit finally joins - it’s joining is catalyed by eIF5 -a GTPase (the GTP bound to the 60s is hydrolysed, this drives the process forward and confirms directionality.)

36
Q

What factors does elongation require?

A

Elongation factors (EFs) (equivalent of eif)

37
Q

Describe the process of eukaryotic elongation in translation

A
  • EF1alpha-GTP forms a ternary complex with the aatRNA
  • EF1a hydrolyses GTP once the anti-codon to codon base pairing is established
  • Peptide bond formation - catalysed by the large rRNA
  • Ribosome translocation, followed by EF2-GTP hydrolysis - an irreversible reaction helping ensure the process has directionality
38
Q

What happens if, during elongation, the correct anit codon-codon pairing is not sensed

A

No hydrolysis by EFa1 occurs (of GTP) so the aatRNA is released and a different, hopefully correct aatRNA will enter the ribosome

39
Q

Where does termination of translation occur

A

at 1 of 3 stop codons

40
Q

What kind of factors are involed in terminatio

A

eukaryotic Release Factors

41
Q

Describe the process of termination

A
  • Ribosome reaches 1 of 3 stop codons
  • release factor eRF1 is in the A site and recognises the stop codon
  • eRF1 promotes the hydrolysis of the bond linking the polypep chain to the tRNA at the P site (helped by the peptidyl transferase centre of the ribosome
  • eRF3 stimulates this process in a GTP dependent manner
  • all components are released
42
Q

What is the special feature of eRF1 that allows it to recognise the stop codon?

A

It mimics tRNA in shape and size
Therefore it loads into the ribosome in tRNA’s place
It has the same Y like stucture, with the 3 branches called 1,2 and 3

43
Q

Which domain of the eRF1 actually recognises the stop codon

A

1

44
Q

Which domain of eRF1 promotes hydrolytic cleavage by the peptidyl transferase centre of the ribosome?

A

2

45
Q

What molecule does eRF1 use to hydrolyse the polypeptidyl-tRNA and release the completed polypeptide chain?

A

H2O.

46
Q

How exactly does eRF1 cause the release of the completed polypeptide chain from tRNA?

A

It (specifically a glutamate residue) coordinates the H2O that carries out the hydrolytic cleavage

  • H2O is used by the peptidyl transferase centre to attack the bond which holds the nacent polypeptide to the tRNA
  • polypep is released
  • This forms a c terminus of newly synthesised proteins
47
Q

What is the often forgotten stage after termination?

A

Recycling

48
Q

What does recycling ensure

A

A high rate of translation

49
Q

What is recycling facilitated by?

A

The circularisation of the mRNA

50
Q

Explain the process of recycling in translation

A
  • The polyA tail is bound by protein, which can interfere with the scaffold protein of the cap binding complex (eIF4G)
  • The 3’end is in close proximity to the start of the mRNA where the cap may be bound (sometimes not, if there’s an IRES instead)
  • This facilitates the passage of ribosomes from the end back to the start - - this is more rapid than allowing the ribosomes back into the cytoplasm
51
Q

How is the efficiency of translation of different mRNA’s compared?

A

More ribosomes, more efficiently translated.

Quantify the no. of ribosomes by testing how fast different mRNAs sediment - more ribosomes, sediment faster

52
Q

What is the word that refers to multiple ribsosomes translating simulataneously

A

polysome

53
Q

What is the main rate limiting step in euk lation

And how is this experimentally shown

A

initiation
manipulating eIFs has marked influence on lation rate
-transform cells by over expressing eIF4G - the fact this results in an increase in speed proves initiation was rate limiting before

54
Q

What is a key regulatory step in initaiatio

A

getting correct mRNAs onto PIC

55
Q

Under some circumstances can elongation be a rate limiting step?
And why for whatever your answer was

A

Yes
Often can improve rate of synthesis by recognising rare codons in polypeptide, and changing them to more common codons
MOre common codons are recognised by tRNAs which are more abundant

56
Q

How could you commercially overproduce a protein?

A

Change codons in sequence to ones recognised by more abundant tRNAs
Due to redundancy in genetic code can do this without changing the sequence
But this is unnatural, and that’s why there was little of the rare tRNA in the first place

Gene to function - Eukaryotes (13 decks)

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