L15: Controlling protein synthesis

Question 1

Protein synthesis is energetically expensive

Answer 1

Activation: each AA requires ATP. ATP -> AMP + 2 PPi (energetically equivalent to 2ATP -> 2ADP + 2Pi)

Elongation: binding of EF-Tu requires GTP: equivalent to 1ATP-> 1ADP + 1Pi. Translocation (EF-G) requires GTP: 1ATP -> 1ADP + 1Pi

Question 2

Ribosomes (eukaryote)

Answer 2

Composed of RNA & protein

80S ribosome: composed of 2 subunits (60S and 40S)

Attached to cytosolic face of ER

Larger than prokaryotic

Question 3

Molecular components for peptide chain initiation

Answer 3

tRNAiMet

mRNA

40S and 60S ribosomal subunits

A set of proteins: eukaryotic initiation factors

GTP

Question 4

Comparisons of molecular components for peptide chain initiation to prokaryotes

Answer 4

Eukaryotic protein synthesis more complex than prokaryotic

Initiator tRNA carries Met and functions only in initiation- it is called tRNAiMet but is formylated

Question 5

Initiation factors in Eukaryotic protein synthesis

Answer 5

Over 12 (more than prokaryotic)

Focus on eIF2: facilitates binding of initiating Met-tRNAMet to 40S ribosomal subunit

Question 6

Post-transcriptional modifications

Answer 6

5’ terminal 7methyl-GTP cap: enhances stability of mRNAs by preventing degradation by 5’exonucleases. Essential for mRNA binding by eukaryotic ribosomes

3’terminal poly(A) tail: poly(A) tail enhances stability of eukaryotic mRNAs. Assists in transport out of nucleus. Involved in initiation of protein synthesis

Question 7

Eukaryotic initiation complex

Answer 7

Post-transcriptional modifications involved: 5’ terminal 7methyl-GTP cap & 3’-terminal poly(A) tail

Circularisation of mRNA at eukaryotic initiation complex

Multiple eukaryotic initiation factors

Poly(A) binding protein

40S subunit

Question 8

Features of peptide chain initiation in eukaryotes

Answer 8

1. Binding of eiFs to 40S subunit
2. Formation of 43S preinitiation complex -> binding of Met-tRNAMet
3. 48S preinitiation complex -> binding of mRNA with eiF
4. Scanning to find first AUG (start) codon
5. Binding of 60S subunit-> GTPase activity -> loss of initiation factors bound to 40S subunit

Question 9

Inhibitors of protein synthesis

Answer 9

Puromycin

Diptheria

Question 10

Puromycin

Answer 10

50S

Binds at A site of both prokaryotic & eukaryotic ribosomes

Accept peptide chain from P site

Terminate protein synthesis

Question 11

Diptheria

Answer 11

Caused by Corynebacterium diptheriae, a facultative anaerobic, Gram +ve

Contagious: spread by physical contact or breathing aerosols

Fatality rates between 5-10%

Largely eradicated by vaccination in industrialised countries

Toxin: ADP-ribosylation occurs on elongation factor 2

Question 12

Regulating activity of protein

Answer 12

1. Initiation of transcription
2. Post-transcriptional processing
3. mRNA stability m
4. Translational regulation
5. Post-translational modification
6. Protein transport
7. Protein degradation

Question 13

Muscles can respond to changes in protein turnover

Answer 13

Hypertrophy. Protein synthesis > protein breakdown

Protein synthesis < protein breakdown. Muscle wasting cachexia and aging

Question 14

Regulation of protein synthesis

Answer 14

Diet

Hormones: IGF-1, growth hormone

Exercise

Mechanism: phosphorylation/dephosphorylation of translational components (dominant mechanism)

Can be activated/inhibited by phosphorylation: 4 IF, 2 elongation factors, ribosomal protein S6

Question 15

eIF2

Answer 15

Facilitates binding of initiating Met-tRNAimet to 40S subunit

Phosphorylation of eIF-2 blocks protein synthesis

alpha-subunit phosphorylation controls function of eIF2

Phosphorylation of eIF-2alpha -> bind to eIF-2B and rendering eIF-2 unavailable for protein synthesis

Question 16

Regulation of translational initiation via phosphorylation of eIF-2alpha

Answer 16

Occurs under number of different conditions: ER stress, AA stress, viral infection, in erythrocytes as consequence of limiting heme