Translation

Question 1

What is translation?

Answer 1

Conversion of mRNA into a polypeptide chain

Question 2

What is the genetic code?

Answer 2

the correspondence between the sequence of the 4 bases in mRNA & the 20 amino acids found in proteins

Question 3

Features of the genetic code?

Answer 3

Triplet code
Non-overlapping
Degenerate
Universal

Question 4

What are synonymous codons?

Answer 4

They all code for the same amino acid

Question 5

What are tRNAs?

Answer 5

The adaptor molecule used for decoding the base sequence of mRNA into the amino acid sequence of proteins

Link between mRNA & the building blocks of proteins

Made by transcription

Question 6

What is the primary structure of a tRNA molecule?

Answer 6

Small nucleic acids of 70-90nts

15 invariant & 8 semi-variant residues in tRNA

Have a 5’ monophosphate (rather than a 5’ triphosphate)

Contain modified bases – aren’t incorporated in the standard process of transcription, they come from the modifications of the standard bases in the post transcriptional process

Question 7

What are the modified bases in the primary structure of tRNA molecules?

Answer 7

Ribothymidine (curly T)
Pseudouridine (fork)
Dihydrouridine (D)
Inosine (I)

Question 8

What is the secondary structure of a tRNA molecule?

Answer 8

D loop – contains dihydrouridine
T loop – contains pseuouridine

Variable arm – 3 to 21 nucleotides
Anti-codon loop – mRNA interacts with the anticodon loop
Amino acid acceptor site

Base pairing in the stems but not in the loops

tRNA 3D structure arises from 9 extra hydrogen bonds

Question 9

What is an aminoacyl tRNA?

Answer 9

A charged tRNA

A tRNA joined to an amino acid

Question 10

How are aminoacl tRNAs formed?

Answer 10

This is a 2 step reaction driven by ATP

1) ATP is added to the carboxyl group of the amino acid to give a high energy intermediate called an aminoacyl adenylate
2) The aminoacyl adenylate reacts with the appropriate uncharged tRNA to give aminoacyl tRNA & AMP

Catalysed by tRNA synthetases

Question 11

Are the tRNA synthetases all the same?

Answer 11

No
Some synthetases join the amino acid to the 2’ hydroxyl & some to the 3’ hydroxyl but once joined the 2 species can spontaneously interconvert

Question 12

What is the process of joining amino acids to tRNA molecules?

Answer 12

Aminoacylation of tRNAs

Aminoacylation drives protein synthesis as the aminoacyl tRNA bond is of higher energy than the peptide bond

Question 13

What are the 2 classes of aminoacyl tRNA synthetases and how are they different?

Answer 13

Class I
Class II

Differ in the way they recognise tRNA – has to recognise the right AA & also the right tRNA

The 2 classes bind different faces of the tRNA molecule

Question 14

How do class I aminoacyl tRNA synthetases bind to the tRNA?

Answer 14

On the underside of the L-shape

Question 15

How do aminoacyl tRNA synthetases proofread?

Answer 15

The acylation site rejects amino acids that are larger than the correct one

The editing site hydrolytic cleaves and rejects amino acids are smaller than correct one

DOUBLE SIEVE MECHANISM

Proofreading improves the fidelity of protein synthesis so that mistakes are made less than once every 10000 amino acids

Question 16

What is the cognate tRNA for serine?

Answer 16

tRNA^Ser

Question 17

What is the cognate aminoacyl tRNA synthesise (uncharged) of serine?

Answer 17

Seryl-tRNA synthetase

Question 18

What is the charged aminoacyl tRNA of serine?

Answer 18

Seryl-tRNA^Ser
or
Ser-tRNA^Ser

Question 19

How do codon-anticodon interactions work?

Answer 19

Codon-anticodon bases pair in an antiparallel fashion

Several features suggest something is missing
• 61 codons but only 20 tRNAs – some tRNAs recognise more than 1 codon
• Base at the 3’ end of the codon isn’t always important

Question 20

What is the Wobble hypothesis by Francis Crick?

Answer 20

The first 2 bases of the codon base pair with the anticodon according to the usual base pairing rules
The base at the 5’ end of the anticodon can form non-standard hydrogen bonds with the base at the 3’ end of the codon

Question 21

What are the 4 wobble base pairs?

Answer 21

I-C
I-U
I-A
G-U

Question 22

How are ribosomes involved in translation?

Answer 22

Ribosomes are the particles on which protein synthesis (translation) takes place

They move along the mRNA & align successive amino acyl tRNAs & amino acids attached one by one to the growing polypeptide chain

Ribosomes are large RIBONUCLEOPRTEIN (RNA-protein) complexes

Question 23

What is the composition of prokaryotic ribosomes?

Answer 23

The 70S structure is the complete prokaryotic ribosome

This can be divided into 2 sub-particles – 50S & 30S

Question 24

What are the 3 stages of translation?

Answer 24

INITIATION
ELONGATION
TERMINATION

Question 25

What does initiation require?

Answer 25

Requires initiation factors 1,2 & 3 and GTP (for energy) The initiator tRNA fMet-tRNA^fMet recognises AUG at the initiator codon but does not recognise methionine codons when they are inside the open reading frame • charged with N-formyl methionine Anticodon more flexible so can recognise GUG as a start codon in E.coli

Question 26

What does elongation require?

Answer 26

Requires elongation factors EF-Tu EF-Ts EF-G & GTP (energy)

Question 27

What does termination require?

Answer 27

Requires a stop codon Requires release factors RF1 or RF2

Question 28

What happens in initiation?

Answer 28

1) The 30S subunit binds the ribosome binding site (RBS) by recognition of the Shine Dalgarno sequence 2) This allows the initiator tRNA (fMet-tRNAfMet) to bind to the start codon AUG 3) This allows the 50S subunit to bind, forming the 70S Initiation Complex on the mRNA, reading to begin translation

Question 29

What are the 3 distinct sites within the ribosome?

Answer 29

The A site – for the acyl group when the amino-acyl tRNA binds The P site – peptidyl where the tRNA attached to the peptide is located The E site – where the uncharged tRNAs get pushed along to

Question 30

How is the shine dalgarno sequence involved in translation?

Answer 30

The Shine Dalgarno sequence (mRNA) is complementary to the 3’ end of the 16S rRNA Base pairing between the Shine Dalgarno sequence and the 16S rRNA properly positions the 30S ribosomal subunit on the mRNA

Question 31

What is the role of initiation factors in the initiation of translation?

Answer 31

1) IF1 and IF3 bind to a free 30S subunit and their job is to get the 30S subunit on to the mRNA and to prevent the 50S subunit binding to it, unless it is first bound to a messenger RNA. 2) IF2 complexed with GTP binds the 30S subunit attaches to a mRNA. 3) A charged initiator tRNA can then bind and base pair with the AUG. 4) IF3 is displaced. 5) A 50S subunit can now bind which displaces IF1 and IF2 and GTP is hydrolysed in this energy consuming step, forming the complete 70S initiation complex.

Question 32

How do elongation factors work?

Answer 32

1) The first step is the delivery of a charged amino acyl tRNA to the A site 2) There is peptide bond formation between the two tRNAs, during which the amino acid joined to the tRNA in the P site loses contact with its tRNA. 3) Then there is translocation where the mRNA is hauled through the ribosome so that the peptidyl tRNA is moved to the peptidyl site.

Question 33

What does the delivery of the charged tRNA in elongation require?

Answer 33

EF-Tu, EF-Ts and GTP EF-Tu is the factor that delivers the charged tRNA to the A site by forming a complex, requiring GTP hydrolysis. However, there is now GDP stuck to the elongation factor. Therefore, EF-Ts recycle the EF-Tu back to the GTP form which can bind the next charged amino acid

Question 34

What does peptide bond formation in elongation require?

Answer 34

Peptide bond formation is catalysed by the ribosome itself so doesn’t need any additional elongation factors

Question 35

What does the translocation step in elongation require?

Answer 35

The translocation step requires EF-G and GTP | No recycling factor is needed in this case because GDP is released at the same time as the phosphate

Question 36

How does termination occur?

Answer 36

Proteins called release factors interact with STOP codons RF1 recognises UAA and UAG RF2 recognises UAA and UGA RF3.GTP (RF3 is bound to GTP) helps RF1 or RF2 carry out termination RRF (ribosome release/re-cycling factor) & EF-G promote dissociation of the ribosome from mRNA

Question 37

What do release factors do?

Answer 37

The release factors direct the ribosome so that the peptide bond is cleaved from the peptidyl tRNA so the polypeptide chain is released. The uncharged tRNA is removed and the ribosome dissociates off the mRNA with the help of RRF, EF-G and GTP hydrolysis