Regulation of Eukaryotic Protein Synthesis

Question 1

Where can malfunctioning happen for transcription?

Answer 1

1) RNA transcript aborts
2) nonfunctional mRNA sequences
3) retention and degradation in nucleus

Question 2

Where can malfunctioning happen for translation?

Answer 2

1) translation blocked

2) RNA degraded

Question 3

How to inhibit protein synthesis specifically eIF2-GDP?

Answer 3

• Double stranded RNA (viral infection)
• Stress
• Nutrient deprivation
• Lack of heme (can’t waste energy on protein synthesis)

^all lead to production of kinase to phosphorylate and inactivate eIF2-GDP and it cannot interact with the exchange factor and no active eIF2-GTP is regenerated (to deliver initiator tRNA)

Question 4

How do phosphorylated eIF2-GDP inhibit?

Answer 4

It binds eIF2B 100x more avidly

Protein synthesis shits down as all eIF2B is bound in a dead-end complex –> protein synthesis diminishes

Question 5

Double stranded RNA

Answer 5

Found in life cycle of HCV

DS RNA as an intermediate form –> stimulates shutting down of host protein synthesis

Long double stranded RNA induces production of interferon

Question 6

Interferon

Answer 6

Stimulates production of kinase that phosphorylates eIF2-GDP

Phosphorylated eIF2-GDP forms suicide complex with exchange factor eIF2B to inhibit protein synthesis in infected cell

Question 7

RNAi

Answer 7

RNA interference can selectively prevent translation and target RNA for degradation

Question 8

PTGS

Answer 8

post-transcriptional gene silencing
agent responsible for this is dsRNA

higher eukaryotes: mi RNA (microRNA)
Therapeutics

Question 9

Lower Eukaryotic PTGS

Answer 9

Double stranded RNA mechanism turns off synthesis of certain proteins in a specific manner

Question 10

Mechanism of RNAi in lower eukaryotes

Answer 10

1) double stranded RNA is cleaved by enzyme Dicer
2) left with 21-25 base double stranded fragments with 2 base overhangs on 3’ end
3) Fragments assemble with RISC (silencing complex with endonuclease activity) thru complimentary sequencing
4) siRNA unwinding so that only one strand (anti-sense) stays in the RISC complex
5) Activated RISC
6) RISC associates with target mRNA thru comp sequences
7) Complex cleaved by Slicer
8) Transcription is prevented

Question 11

Mechanism of RNAi in higher eukaryotes

Answer 11

Do not synthesize long dsRNA
Synthetic siRNA can be introduced to target specific mRNAs for degradation
Then the siRNA is processed same way as lower eukaryotes

Question 12

miRNA

Answer 12

Naturally occurring small silencing RNA in mammals

Precursor: Long hairpin structures in the nucleus synthesized by Pol II and processed by Drosha to form pre-miRNA

Transported to the cytoplasm and processed by Dicer–> unwound –> RISC retains antisense strand

miRNA + RISC –> bind to mRNA 3’ untranslated regions (imperfect pairing) to inhibit translation (can also lead to mRNA degradation if perfect pairing)

Mediates control of proliferation, cell death, embryonic development, and patterning

Question 13

How does RISC work in miRNA

Answer 13

1) deadenylation: removes polyA tail on RNA
2) decapping: sequestered in the P body
3) Target mRNA degradation

*no slicing like with siRNA but miRNA binding to 3’ untranslated region causes enzymes to remove poly A tail *

Question 14

Miraversin for HCV treatment

Answer 14

Blocks the actions of Drosha and Dicer for miRNA

Therefore, mature miRNA is never formed from pre-miRNA and then thus HCV RNA will not be made

Question 15

Gene Therapy

Answer 15

Restore gene where missing/mutant

Suppress expression of mutant/dysregulated gene