CMB2001/L09 Post-transcriptional Control IIa

Question 1

What is the composition of a ribosome?

Answer 1

1/3 protein
2/3 mRNA

Question 2

Describe tRNA charging (aminoacylation). (3)

Answer 2

Amino acid activation through nucleophilic attack (forming aa-AMP)
Formation of aminoacyl-tRNA (OH group of adenine 76 of tRNA attacks carbonyl carbon of aminoacyl-adenylate)
Forms aminoacyl-tRNA and AMP

Question 3

Where in the ribosome do peptide bonds form?

Answer 3

P - peptidyl site
A - aminoacyl site

Question 4

Give the 5 steps of translation elongation.

Answer 4

Aminoacyl-tRNA binding
Peptide bond formation
Translocation
GTP hydrolysis
Repeat

Question 5

Give 2 features specific to eukaryotic mRNAs.

Answer 5

Cap
PolyA tail

Question 6

Describe eukaryotic translation initiation. (5)

Answer 6

Small subunit binds CAP
Scans to first AUG for Met
Met-tRNA positioned
Large subunits joins
Transition to elongation

Question 7

Why is mRNA circularised during initiation of translation?

Answer 7

Monitors integrity of mRNA
Brings ribosomes ending translation close to start site
Several other key translation factors

Question 8

Give 3 proteins required for mRNA circularisation.

Answer 8

elF4E
G
PAB

Question 9

What is the role of elF4E?

Answer 9

M7G binding

Question 10

What is the role of elF4G?

Answer 10

Binds elF4E, a, 3, PABP

Question 11

What is the role of elF4A?

Answer 11

ATPase
RNA helicase

Question 12

What is the 43S pre-initiation complex?

Answer 12

Small subunits primed for associating with mRNA

Question 13

What is the role of elF1A?

Answer 13

80S dissociation
Met-tRNA binding to 40S

Question 14

What is the role of elF3?

Answer 14

80S dissociation
Binds many other elFs

Question 15

What is the role of elF1?

Answer 15

AUG recognition

Question 16

What is the role of elF2?

Answer 16

GTPase
Met-tRNA binding
Binds elF5

Question 17

What is the role of elF5?

Answer 17

Stimulates elF2 GTPase GAP for elF2

Question 18

What is needed for 43S association?

Answer 18

Interaction of elF3 and elF4G
RNA unwinding (elF4F unwinds cap-proximal sequence)

Question 19

What needs to occur to generate the ternary complex for further initiation events?

Answer 19

Recycling of elF2

Question 20

What is the role of elF5B?

Answer 20

GTPase
Promotes subunit joining

Question 21

Give a role of elF2B in the elF2 cycle.

Answer 21

Governs level of elF2-GTP (therefore initiation rate)

Question 22

Explain regulation of elF2B. (3)

Answer 22

Lower level than elF2
Down-regulated in response to stresses (viral infection, amino acid deprivation, ER stress)
Regulated through phosphorylation of elF2 (competitive inhibitor of elF2B)

Question 23

Describe the structure of elF2.

Answer 23

3 subunits (a, B, y)
a phosphorylated on Ser51 by PKR, PERK, GCN2, HRI
B binds elF2B, elF5
elF2y is a GTPase, binds Met-tRNA

Question 24

How is PKR activated?

Answer 24

By double stranded RNA (viral infection)

Question 25

What is PERK?

Answer 25

Mediator of unfolded protein response (ER stress)

Question 26

What is GCN2?

Answer 26

Regulator of translation in response to amino acid availability

Question 27

What is HRI?

Answer 27

Links globin availability to protein synthesis Critical for RBC biogenesis

Question 28

What happens to elF2 kinases under stimulatory conditions?

Answer 28

Dimerise Autophosphorylate and act on substrate

Question 29

Describe regulation of PKR.

Answer 29

Typically low expression Increases when cells exposed to interferons (viral infection) Binds dsRNA

Question 30

Describe iron response elements (IREs). Where are the found? What are they bound by?

Answer 30

Hairpin loops with conserved loop sequence Bulge in stem Found in 5' or 3' UTRs of iron-regulated mRNAs Bound by IRP1&2

Question 31

Describe how low iron affects mRNA stability and translation.

Answer 31

IRP1&2 bind to IREs in mRNA Inhibit translation by blocking ribosomal access or destabilising mRNA Binding of IRPs protects from degradation

Question 32

Describe how high iron levels affect mRNA translation and stability.

Answer 32

Iron binds to IRP1 to reduce affinity for IREs, promoting ribosomal access and translation Absence of bound IRP to mRNA promotes degradation

Question 33

Why is IRP1 described as a bifunctional protein?

Answer 33

With or without iron, has different functions -Fe -> C-aconitase interconverts citrate and isocitrate +Fe -> IRE1