W2 Protein synthesis

Question 1

What is the methyl cap?

Answer 1

During transcription 5’-5’ linked 7-methylguanylate and next two nucleotides have methylated 2’-OH on ribose moieties added

Question 2

Speed/location of PS

Answer 2

W/in cleft b/een RS units, fast (20 AAs per s + actin every 20 s)

Question 3

80S structure

Answer 3

• Peptidyl binding site on 60S
• Aminoacyl tRNA binding site on 40S
• 40S = 18S rRNA (1900 nucleotides) ~ 33 proteins
• 60S = three RNAs 5S, 5.8S and 28S (120, 160 & 4700
  nucleotides) ~ 49 proteins
• RS ⅔ RNA + ⅓ protein

Question 4

Initiation

Answer 4

• RS assembled w/mRNA, first initiator tRNA = aminoacyl tRNA (methionine-tRNAi = binds to P site instead of A on tRNA) - met-tRNAi brought by eiF2
• 40S assembles on cap
• eiF3 binds + blocks E site on 40S (eiF3 prevents 60S from prematurely binding)
• eiF3 + other initiation factors bound to 40S binds eiF2-GTP = ternary complex
• Initiator tRNA carries methionine (formyl-methionine in bacteria)
• eiF4 promotes 43S to 5’ cap (and binds) + PAB (PolyA binding protein) binds polyA tail
• Scanning of 5’ UTR + AUG - 43S scans 5’ UTR to localise AUG (48S complex)
• mRNA attaches to 40S = preinitiation complex = 48S
• Hydrolysis of GTP by eiF5 (due to AUG pairing w/met-tRNAi eiF5 activating protein for GTPase) and release of initiation factors eiF2 and eiF3
• Joining of 60S subunit due to eiF1A + eiF5B + release of energy by GTP
• Met-tRNAi associates with P site and tRNA enters A site with eEF1
• Peptidyl transferase catalyses peptide bond
• Assembly of 80S

Question 5

Translocation

Answer 5

Movement of CS segment from one position in genome to another (could be w/in same CS)

Question 6

Elongation

Answer 6

• aa-tRNA (charged) binds to A on 30S
• Ternary complexes of eF1A:aa-tRNA:GTP enter RS + allow tRNA to pair w/A site codon
• Peptidyl transferase catalyses formation of PP bond b/een incoming AA + peptide on P site
• Elongation factors EF1 involved in energy (GTP) dependent tRNA binding to ribosome
• Formation of peptide bond and EF2 dependant translocation of the ribosome along the message occupying P & E site
• Release of spent tRNA (which is now deacylated) from E site

Question 7

Polysomes

Answer 7

Initiation occurring many times

Question 8

Termination

Answer 8

• Hydrolysis of terminal peptidyl tRNA bond and dissociation of ribosome
• Ribosome translocates to stop codon (UAA,UGA or UAG) and binds release factor at A site
• Release factor = eRF1 (also known as TB3-1)

Question 9

Energy requirements

Answer 9

Charging tRNAs w/AAs = 1 ATP/aa

Initiation (assembly of RS on mRNA w/first aa-tRNA + scanning) = GTP + nATP

Elongation (addition of AAs one at a time) = 2 GTP/aa

Termination (release of PP) = GTP

Question 10

70S

Answer 10

Large/small = 50S/30S

Large = 23S + 5S RNA ~ 31 proteins

Small = 16S ~ 21 proteins

Question 11

Streptomycin

Answer 11

Binds to small ribosomal unit + inhibition of initiation, misreading of genetic code

Question 12

Tetracylcines

Answer 12

Binds to small ribosomal unit + inhibition of aa-tRNA binding to RS

Question 13

Chloramphenicol

Answer 13

Binds to large ribosomal unit + inhibition of peptidyl transferase activity

Question 14

Erythromycin

Answer 14

Binds to large ribosomal unit + inhibition of translocation

Question 15

Neomycins

Answer 15

Binds to multiple sites (30S) + several effects (inhibits translation)
High binding affinity for PIP2