Lecture 40: Protein Synthesis Control

Question 1

Methods of post-transcriptional gene regulation in eukaryotes

Answer 1

1. Initiation factor control
2. RNA interference

Question 2

Guanine Nucleotide Exchange Factor

Answer 2

Important to catalyze regeneration of eIF2-GDP to eIF2-GTP, reactivating eIF2.

Question 3

Regulation of GEF for eIF2 reactivation

Answer 3

Stress induces global lower protein synthesis rate.
Kinase production phosphorylates eIF2-GDP creating a dead-end complex that can’t be regenerated, inhibiting protein synthesis e.g. in the presence of dsRNA

Question 4

dsRNA in eukaryotes

Answer 4

dsRNA in eukaryotes must be a viral signal; doesn’t exist normally. Results in interferon production increasing kinase production to lower protein synthesis. eIF2-GDP will form a suicide complex w/ eIF2B exchange factor

Question 5

RNAi

Answer 5

RNA interference allows specific control (not global) of protein synthesis

Question 6

Types of RNAi

Answer 6

Lower eukaryotes: long dsRNA (from RNA dependent RNA polymerase)
Higher eukaryotes: miRNA
Therapeutics/experiments: siRNA

Question 7

Lower eukaryotic RNAi

Answer 7

1. Produced dsRNA dicer cleavage to siRNA
2. siRNA assoc. w/ RISC
3. RISC retains antisense strain (complementary), sense removed
4. Antisense siRNA binds target mRNA w/ perfect base pairing at coding region
5. Slicer (argonaute) cleaves siRNA-mRNA complex, degrading mRNA

Question 8

RISC

Answer 8

RNA induced silencing complex; mediates silencing of target mRNA through base pairing

Question 9

High eukaryotic RNAi mechanism

Answer 9

No dsRNA synthesized. Same dicer/slicer w/ miRNA or exogenous siRNA
1. Hairpin miRNA precursor synth. in nucleus by RNA poly III
2. Drosha processing of hairpin precursor -> pre-miRNA
3. Pre-miRNA transport to cytoplasm; dicer process, antisense retained RISC
4. IMPERFECT pairing miRNA to 3’ UTR of target mRNA, protein repress. by translat. inhib.
4a. Perfect pairing -> slicer degradation instead
5. Repression sequestered to P-body of cell

Question 10

HCV miR-122

Answer 10

miR-122 stabilizes HCV RNA 5’ UTR, allowing susceptibility to HCV infection. However, low miR-122 is cancer risk

Question 11

Where are nuclear encoded proteins synthesized?

Answer 11

All are synthesized on cytoplasmic ribosomes, either free or rough ER bound

Question 12

Nuclear encoded proteins synthesized by free cytoplasmic ribosomes

Answer 12

1. Cytoplasmic proteins
2. Mitochondrial proteins
3. Nuclear proteins

Question 13

Nuclear encoded proteins synthesized by rough ER bound ribosomes

Answer 13

1. Cell membrane proteins
2. Secreted proteins
3. Lysosomal proteins

Question 14

How is cell localization encoded in a protein?

Answer 14

Via localization signals in primary structure.
- Cytoplasm: no signal
- Mitochondrial: amphipathic pre-seq.
- Nuclear: short pos. seq. in middle of protein
- Membrane/secretory: signal seq. w/ hydrophobic core
- Lysosomal: mannose-6-Pi

Question 15

Co-translational ER membrane insertion of secretory, membrane, lysosomal proteins

Answer 15

1. Signal Recognition Particle recognizes signal peptide (RNA + protein components)
2. Translation stops
3. Rough ER SRP receptor opens ER membrane
4. Protein threaded into ER membrane co-translationally

Question 16

Secretory/lysosomal protein co-translational membrane insertion

Answer 16

Membrane-signal peptide is cleaved by signal peptidase, full protein ends up in ER lumen

Question 17

Membrane protein co-translational membrane insertion

Answer 17

Stop-transfer seq. further in protein reaches ER membrane -> signal peptidase cleaves start transfer signal seq.; start-stop allows embedding multiple parts of protein in membrane

Question 18

ER protein glycosylation

Answer 18

N (Asparagine) glycosylation produces mannose-6-Pi signal for lysosomal proteins; 1 chunk of sugar added to Asn of Asn-X-Ser/Thr

Question 19

Producing mannose-6-Pi signal for lysosomal proteins

Answer 19

Mannose can’t be phosphorylated directly
1. Glucose lost in ER
2. Transport to Golgi
3. Addition of Pi-sugar (P-Glc-NAc)
4. Removal of sugar leaves mannose-6-Pi behind
5. M6P receptors in trans Golgi allow vesicle to bud off

Question 20

Mechanisms behind lysosomal storage diseases

Answer 20

1. Defect in lysosomal enzyme itself
2. Sugar transfer defect (broken localization signal)

Question 21

Common characteristic of LSDs

Answer 21

All lysosomal storage diseases result in difficulty synthesizing sphingosine e.g. Fabry disease