Protein synthesis - Basic Principles II

Question 1

Explain Completion of Initiation and start of elongation (step 3) in prokaryotes ?

Answer 1

IF2-GTP-femet-tRNA promotes association of the 50S subunit.GTP is then hydrolysed and IF2 is ejected

Question 2

What then delivers the next aminoacyl tRNA ?

Answer 2

EF-TU-GTP delivers the next aminoacyl tRNA

Question 3

Explain how peptide bond formation occurs ?

Answer 3

• Base pairing leads to EF-TU hydrolysing GTP, which is then ejected
• Peptidyl transferase activity makes a peptide bond between the amino acids

Question 4

What happens when EF-G and GTP bind ?

Answer 4

EF-G-GTP binds, GTP is hydrolysed (causes the ribosome to slide along the mRNA) and the ribosome slides and places the next codon in the A-site

Question 5

Where does ejection of tRNA occur ?

Answer 5

• Ejection of tRNA occurs at the E-site

- Elongation continues until we reach a stop codon

Question 6

Explain Termination (step 4) ?

Answer 6

• RF1/RF2 similar in shape to tRNA and recognises stop codons
• RF3 monitors the correct codon interaction with RF1/2. This causes hydrolysis of its GTP, thus stimulating RF1/2 to stimulate the petidyltransferase activity of the ribosome causing release of the polypeptide chain.
• The free tRNA is released together with the polypeptide and the RF and ribosome separate. IFs bind to restart the process

Question 7

What termination codons do RF1 and RF2 recognise ?

Answer 7

• RF1 recognises the termination codons UAA and UAG

- RF2 recognises UAA and UGA

Question 8

What does eEF1 alpha have the same function as ?

Answer 8

eEF1 alpha same function as EF-TU (delivers aminoacyl-tRNA)

Question 9

What does eEF2 have the same function as ?

Answer 9

eEF2 same function as EF-G (stimulates translocation of the ribosome)

Question 10

What does eRF1 have the same function as ?

Answer 10

eRF1 same function as RF1/2, but recognises all stop codons

Question 11

What does eRF3 have the same function as ?

Answer 11

eRF3 same function as RF3

Question 12

What is a typical chaperone action ?

Answer 12

1. Exposed hydrophobics bound by chaperone
2. Reduces concentration of exposed hydrophobic residues
3. Reduces IRREVERSIBLE aggregation of hydrophobic residues

Question 13

Explain Refolding ?

Answer 13

1. Cycles of binding and release by chaperone
2. Local regions of polypeptide begin to fold (nucleation)
3. Folded region no longer bound by chaperone
4. Eventually the protein folds (specific pathway)

Question 14

For refolding, what is the concentration of the solvent?

Answer 14

At all times the concentration of solvent exposed hydrophobic residues is kept low

Question 15

What is the function of a trigger factor ?

Answer 15

Trigger Factor (TF) is an ATP-independent chaperone protein that assists in folding and prevents misfolding

Question 16

Fast on and off rates lead to?

Answer 16

Localised folding and eventual release from trigger factor into a fully folded protein

Question 17

Failure to fold means ?

Answer 17

Failure to fold means transfer to Hsp70 system

Question 18

What is Nascent associated complex (NAC) ?

Answer 18

Similar to trigger factor but not homologues

Question 19

What is Ribosome associated complex (RAC) ?

Answer 19

A Hsp70/Hsp40 based chaperone system

Question 20

Explain the role of chaperones in ER import ?

Answer 20

1. Post-translational secretion (across E. coli cell membrane)
2. Co-translational secretion (translocation). Basic components for E.coli secretion and ER import (SRP, SRP-R and Spase)
3. Import into the ER: Add in a ratchet based Hsp70 system