Lecture 7- Translation and Protein Synthesis

Question 1

In which direction is mRNA translated?

Answer 1

5’ –> 3’ or N terminus –> C terminus

Question 2

Define the term codon

Answer 2

A sequence of three nucleotides found on mRNA which code for a specific amino acid

Question 3

What is the UTR and suggest it’s function

Answer 3

UTR= untranslated region

1) enhance translation
2) mRNA stability

Question 4

How many different codons and amino acids are there?

Answer 4

Codons- 64 (4^3)
Amino acids -20
–> universal code is degenerate= amino acids are coded by more than one triplet codon

Question 5

What is the start codon and amino acid?

Answer 5

Methionine = AUG

Question 6

What are the three different stop codons?

Answer 6

UAA, UAG, UGA

Question 7

5’ CGC UGA AUG CAU GGG CGC CUG AAG GUA AAG ACG UCG GCU GAA GAG CAG GCA GAG GCC AAA AGG CAG UGA GCC 3’

In this sequence where would you start and stop translation and why?

Answer 7

• ribosomes start scanning at 5’ end
• start at first AUG start codon
• continues in frame reading triplet codons
• stops scanning at first in frame stop codon (UGA)

Question 8

What are the sizes of prokaryotic and eukaryotic ribosomes? Also state the sizes of their subunits

Answer 8

Prokaryotic= 70S (50S + 30S)
Eukaryotic= 80S (60S + 40S)
S= Svedberg, the unit of sedimentation in a centrifuge

Question 9

Explain how the fidelity of the genetic code is maintained

Answer 9

Aminoacyl tRNAs
Specific tRNA for the amino acid that it carries on the 3’ receptor
Anticodon on stem hydrogen bonds with the codon on mRNA

Question 10

Explain how amino acid tRNA synthetase is charged….

Answer 10

Specific tRNA synthetase for specific amino acid

• specific amino acid binds to the enzyme
• ATP binds to the enzyme
• covalent bond forms between AMP and amino acid
• specific tRNA binds to enzyme, anticodon and 3’ receptor are specific to enzyme
• AMP dissociates from enzyme and phosphodiester bond forms between amino acid and tRNA = charged
• amino acyl tRNA dissociates from enzyme

Question 11

What happens during the initiation steps of translation?

Answer 11

1) The ribosomal subunits dissociate (40S + 60S)
2) Assembly of pre-initiation complex
- EIF4E and EIF4G bind with the 5’ cap which is recognised by the 40S/ Met-tRNA/eIF2
- 40S= mRNA and tRNA recognition
3) Binding of mRNA to pre-initiation complex. Initiator Met binding sets frame
4) Binding of 60S subunit. GTP–> GDP + Pi . Met tRNA binds to ‘peptidyl’ P site on ribosome
5) eIF2 and GDP dissociate

Question 12

Describe the steps involved in the ‘elongation’ phase

Answer 12

1) Binding of tRNA to amino acyl (A) site of ribosome
2) Catalysis of peptide bond by peptidyl transferase on 60S
3) Translocation of peptidyl tRNA from A to P with help of elongation factors to help move ribosome along mRNA
4) repeat

Question 13

Describe the steps involved in the ‘termination’ phase

Answer 13

1) Recognition of the step codon.
- vacant A site recognised
- Release factors not tRNA bind to stop codon
2) Release of peptide chain.
- Peptidyl transferase catalyses the transfer of the completed protein chain to water
- protein is released from ribosome

Question 14

What are polyribosomes?

Answer 14

One ribosome does not translate one mRNA at a time but multiple ribosomes at a time to increase the rate of protein synthesis

Question 15

Why do many antibiotics target protein synthesis?

Answer 15

Because we can exploit the differences between prokaryotic and eukaryotic ribosome and translation factors

Question 16

Name some antibiotics and the stages of translation they interfere with

Answer 16

1) Streptomycin- inhibits initiation
2) Tetracycline- Inhibits aa-tRNA binding
3) Erythromycin- inhibits translocation
4) Chloramphenicol- inhibits peptide transferase
5) Puromycin- terminates elongation prematurely

Question 17

Where does protein translation take place?

Answer 17

In the cytoplasm (+some in mitochondria)

Question 18

What do transmembrane/ secretory proteins have?

Answer 18

Signal sequences

• first 20-24 aa
• N-terminus of pp
• mainly hydrophobic aa’s which like to sit within the lipid bilayer

Question 19

Describe the process of synthesising proteins on the RER

Answer 19

1) SS is recognised by a protein-RNA complex (signal recognition particle)
- SRP binds to ss
- translation is halted
2) SRP binds to SRP receptor on RER surface
- Translation resumes
3) Translocation of protein chain into RER lumen
- as protein is being translated SRP-receptor binding triggers assembly of protein channel within RER membrane
- PP chain enters lumen across the channel
4) SS is cleaved by signal peptidase once protein has entered. Protein is folded. SS degrades in lumen.

Question 20

What is the difference between the synthesis of transmembrane and secretory proteins?

Answer 20

Transmembrane have an extra hydrophobic sequence at the C-terminus holding them in the membrane

Question 21

List some of the post-transitional modifications that could be made.

Answer 21

• disulphide bond formation
• proteolytic cleavage
• addition of carbohydrate
• addition of phosphate
• addition of lipid groups
• hydroxylation

Question 22

Describe the post translational modifications that occur to produce insulin

Answer 22

• synthesised on RER ribosomes PREPROINSULIN
  1) SS is cleaved and degraded
  2) Disulphide bonds between two cysteine residues. 3 disulphide bonds form + folding –> PROINSULIN
  3) Proteolytically cleaved in 2 positions which releases C chain.
  4) Packaged into secretory vesicles. Has A and B chains held together by 3 x S-S bonds