lecture 9 Flashcards
What is a ribosome made up of?
~1/3 protein
~2/3 RNA
How do tRNA bind?
Anticodon binds to codon
3’ end, CC end
What reaction does aminoacyl-tRNA synthetases catalyse?
1) Amino acid activation - amino acid and ATP bind catalytic site, nucleophilic attack by α-carboxylic acid oxygen yielding aminoacyl-adenylate (aa-AMP)
2) Hydroxyl group of adenine 76 of tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP
How is a peptide bond formed between aminoacylated tRNA and polypeptide?
Takes place on polypeptide chain site of ribosome
The aminoacylated tRNA binding site before the polypeptide chain site
The exit site after the polypeptide chain site to allow mRNA to leave ribosome
Are tRNAs present on the aminoacylated tRNA binding site and polypeptide chain site?
Yes
eg: the expanding polypeptide chain is attached the the P-site tRNA
How does translation elongation occur?
mRNA moves along three bases to introduce new tRNA to ribosome initiated by elongation factor
What is peptide bond formation catalysed by?
the ribosome
What does eukaryotic translation initiation depend on?
The 5’ cap on the mRNA
Small subunit binds the cap, moves (scans) to the first AUG, encoding the initiating methionine of the protein
What is the Kozak consensus sequence?
5’-ACCAUGG-3’
The most frequently found sequence around the AUG in eukaryotic mRNA
Why is mRNA circularised during translation initiation?
Monitors integrity of mRNA
Brings ribosomes ending translation close to the start site
Brings proteins ending translation close to the cap
Key translation factors bind to it also
What are the proteins needed for mRNA circularisation?
elF4E, G, PAB
What is the 43S pre-initiation complex?
Small subunit primed for associating with mRNA
Preparing small subunit (40S) for translation
EIF3 multifactorial
Prevent 40S from binding to 60S to keep in cytoplasm for translation
What is needed for 43S association?
Interactions - elF3 with elF4G
RNA unwinding: most 5’ UTRs has at least some structure - elF4F (4A) unwinds cap-proximal sequence
What is the 48S pre-initiation complex?
Small subunit associated with the AUG codon
5’ proximal AUG is used unless very close to cap
Consensus GCC(A/G)CCAUGG inserted before natural AUG meaning new AUG is used
Mutation of initiator tRNA anticodon (to 3’-UCC-5’) results in use of complementary codon (AGG)
Requires elF4F
What are the main parts of elongation translation?
-elF4F complex binds cap
-Small subunit recruited to the cap
-then scans along until it finds the start AUG
-large subunit joins
-Translocation to move the tRNAs and mRNA through the ribosome
-reach termination codon, translations stops and the ribosome dissociates
What are the key regulation points of translation?
Formation of elF4F
43S Binding
Function of elF2B/ Ternary complex formation
How is elF2 regulated?
Activated by elF2B
Multiple protein kinases phosphorylate a single serine
This inhibits the GEF activity of eIF2B
What is elF2Bs function ?
Regulates protein synthesis
It reactivates the G protein eIF2, which, in its GTP-bound form, binds initiator methionyl-tRNA (Met-tRNAMeti) to form GTP-eIF2 ternary complex (TC)
The TC is delivered to the 40S small ribosomal subunit before mRNA binding
How is elF2B different to elF2?
elF2B is present at a much lower level
elF2B activity governs level of active elF2-GTP and thus overall initiation rate
How is elF2B regulated?
Through phosphorylation of elF2, which becomes a competitive inhibitor of elF2B
What is a consequences of elF2 acting as a competitive inhibitor for elF2B?
generation of ternary complex is impaired, translation initiation of mRNA is reduced
What cause elF2B to be down-regulated?
In response to stresses, such a viral infection, amino-acid deprivation, endoplasmic reticulum stress
What proteins regulate the elF2 cycle?
elF2α - phosphorylated on Ser51 by PKR, PERK, GCN2, HRI
elF2β - binds elF2B, elF5
elF2γ - GTPase, Met-tRNA
What is the key regulatory step in the elF2 cycle?
Since elF2 > elF2B, a small amount of elF2 phosphorylation can decrease ternary complex levels substantially
