CMB2001/L09 Post-transcriptional Control IIa Flashcards
What is the composition of a ribosome?
1/3 protein
2/3 mRNA
Describe tRNA charging (aminoacylation). (3)
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
Where in the ribosome do peptide bonds form?
P - peptidyl site
A - aminoacyl site
Give the 5 steps of translation elongation.
Aminoacyl-tRNA binding
Peptide bond formation
Translocation
GTP hydrolysis
Repeat
Give 2 features specific to eukaryotic mRNAs.
Cap
PolyA tail
Describe eukaryotic translation initiation. (5)
Small subunit binds CAP
Scans to first AUG for Met
Met-tRNA positioned
Large subunits joins
Transition to elongation
Why is mRNA circularised during initiation of translation?
Monitors integrity of mRNA
Brings ribosomes ending translation close to start site
Several other key translation factors
Give 3 proteins required for mRNA circularisation.
elF4E
G
PAB
What is the role of elF4E?
M7G binding
What is the role of elF4G?
Binds elF4E, a, 3, PABP
What is the role of elF4A?
ATPase
RNA helicase
What is the 43S pre-initiation complex?
Small subunits primed for associating with mRNA
What is the role of elF1A?
80S dissociation
Met-tRNA binding to 40S
What is the role of elF3?
80S dissociation
Binds many other elFs
What is the role of elF1?
AUG recognition
What is the role of elF2?
GTPase
Met-tRNA binding
Binds elF5
What is the role of elF5?
Stimulates elF2 GTPase GAP for elF2
What is needed for 43S association?
Interaction of elF3 and elF4G
RNA unwinding (elF4F unwinds cap-proximal sequence)
What needs to occur to generate the ternary complex for further initiation events?
Recycling of elF2
What is the role of elF5B?
GTPase
Promotes subunit joining
Give a role of elF2B in the elF2 cycle.
Governs level of elF2-GTP (therefore initiation rate)
Explain regulation of elF2B. (3)
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)
Describe the structure of elF2.
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
How is PKR activated?
By double stranded RNA (viral infection)
What is PERK?
Mediator of unfolded protein response (ER stress)
What is GCN2?
Regulator of translation in response to amino acid availability
What is HRI?
Links globin availability to protein synthesis
Critical for RBC biogenesis
What happens to elF2 kinases under stimulatory conditions?
Dimerise
Autophosphorylate and act on substrate
Describe regulation of PKR.
Typically low expression
Increases when cells exposed to interferons (viral infection)
Binds dsRNA
Describe iron response elements (IREs). Where are the found? What are they bound by?
Hairpin loops with conserved loop sequence
Bulge in stem
Found in 5’ or 3’ UTRs of iron-regulated mRNAs
Bound by IRP1&2
Describe how low iron affects mRNA stability and translation.
IRP1&2 bind to IREs in mRNA
Inhibit translation by blocking ribosomal access or destabilising mRNA
Binding of IRPs protects from degradation
Describe how high iron levels affect mRNA translation and stability.
Iron binds to IRP1 to reduce affinity for IREs, promoting ribosomal access and translation
Absence of bound IRP to mRNA promotes degradation
Why is IRP1 described as a bifunctional protein?
With or without iron, has different functions
-Fe -> C-aconitase interconverts citrate and isocitrate
+Fe -> IRE1
