Protein synthesis

Question 1

Structure of an eucaryotic mRNA?

Answer 1

m7G = methylated guanine cap “head”
mRNA
Poly-A Binding Protein “tail”

Question 2

Overview of host cell protein synthesis

Answer 2

1. Eucaryotic initiation factor complex (eIF4F) recognises 5’ cap structure
2. eIF4F recruits the 40S ribosomal subunit
3. 40S scans the mRNA until AUG, then recruits 60S

Protein translation!

Question 3

Role of 4EBP1?

Answer 3

Negative regulation of initiation:

4EBP1 binds to eIF4E to stop eIF4F complex from forming - REMINDER THAT IT’S A COMPLEX REGULATOR

Question 4

What is eIF4F complex made of?

Answer 4

eIF4E, eIF4G, eIF4A

Question 5

How does viruses interact with eIF4G

Answer 5

• NS1 interacts with 5’end of viral RNA
• NS1 interacts with both PABP and eIF4G
• PABP and eIF4G recruited to virus RNA
• Viral translation enhanced

100K interacts with eIF4G and displaces MNK1
Dephosphorylation of eIF4E
Block to translation initiation

Rotavirus nsp3 interacts with eIF4G and displaces PABP
Block to translation

Question 6

What is cap snaping?

Answer 6

Snatching the cap means the eIF4F complex is not recruited –> stops translation of host cell proteins
Cap on viral genome instead –> starts translating viral proteins

Question 7

Regulation of translational initiation by eIF2?

Answer 7

*Scan for AUG
Release eIFs + Recruit 60S

eIF2 becomes eIF2B by converting GTP –> GDP

RECYCLE!

Question 8

What does Phosphorylate eIF2 alpha do to the regulation of Regulation of eIF2?

Answer 8

Phosphorylate eIF2 alpha stops translation

Question 9

Different types of Ser51P kinases?

Answer 9

GCN1
Responds to amino acid starvation or UV

HRI
Responds to haem deprivation

PERK
Responds to endoplasmic reticulum stress

PKR
Responds to dsRNA

Question 10

Role of Protein kinase R (PKR)?

Answer 10

Expression is stimulated by IFN but activation requires binding to dsRNA
Autophosphorylates and then phosphorylates eIF2a
Inhibits initiation of translation

Question 11

Viral inhibition of PKR?

Answer 11

*Picornaviruses – activate cellular pathway to degrade PKR
May involve lysosomal degradation

• Some produce double stranded RNA which sequester it.
• Some prevent the phosphorylation of Eif-2A + IKB

Question 12

How do viruses ensure translation of their proteins?

Answer 12

• Many viruses have an Internal Ribosome Entry Site (IRES)
• Mediate cap-independent initiation of translation
• Reduced requirement for initiation factors
• 40S ribosome will bind directly to the IRES

Question 13

How does Picornavirus undergo protein synthesis?

Answer 13

40S binds directly to IRES region
Ribosome scans to AUG
60S recruited
Protein translation

Question 14

How many classes of IRES?

Answer 14

4 - each numbered 1-4

Question 15

How does Poliovirus IRES work?

Answer 15

• Poliovirus IRES binds to C-terminal domain of eIF4G
• Requires eIF5, eIF2 and Met-tRNA
• Stimulated by eIF1 and eIF1A

Question 16

How does HCV IRES work?

Answer 16

HCV IRES binds to eIF3 and 40S ribosome
Requires eIF5 and Met-tRNA
No requirement for eIF2 or eIF4
HCV IRES binds to eIF3 (RNA-protein interaction) and 40S ribosome (RNA-RNA interaction)

Question 17

How does Calicivirus protein synthesis work even though it has no IRES?

Answer 17

VPg binds to eIF4G and eIF3
 May be other interactions (VPg homology to eIF1A) 
 Recruits 40S ribosome complex
 Orf1 AUG only 10 nucleotides from 5’end
 No ribosome scanning
 VPg is a protein cap analog