Lecture 9. The Mechanics of Translation Flashcards
What are the key differences between eukaryotic and prokaryotic mRNA?
Have different initiation and termination regions
Eukaryotic mRNA has a 5’ cap ahead of the start codon, prokaryotic mRNA has a ribosome binding site (RBS)
Eukaryotic mRNA has a poly(A) tail at the 3’ end, prokaryotic mRNA does not
What is the start signal in prokaryote mRNA?
AUG (or sometimes GUG) preceded by several bases that pair with 16S rRNA
The purine-rich region, called the Shine-Dalgarno sequence is complementary to the
initiator sites of mRNA
What are the two types of tRNAmet (targets methionine/Met) in E. coli?
tRNAfmet : Met residues attached to this are formylated
Initiate polypeptide chains only
Recognises AUG and GUG (GUG internally codes for valine)
Can only be used in the initiation of translation
tRNAmmet : Met residues are only attached, not formylated
Recognises the codon AUG only
Used as a source of internal met residues
Can only be sued during the process of extension
In the prokaryotic system, what is required for protein synthesis?
Initiation of protein synthesis in bacteria require free 30S subunits and three proteins (factors) that are necessary for initiation. Termed IF-1, IF-2 and IF-3
How is translation initiated in the 30S subunit of prokaryotic ribosomes?
IF-3: binds to the 30S subunit and prevents association with the 50S subunit
IF-1 binds near the A site therefore directs fmet-tRNA to the P site
IF-2 reacts with fmet-tRNA and GTP to form a ternary complex IF-2-GTP-fmet-tRNA
Delivers the ternary complex and mRNA to the partial P site in the 30S subunit-mRNA complex
Triggers GTP hydrolysis when the 50S subunit joins the complex
It does not recognise met-tRNA or any amino acid (aa) tRNA used for elongation
How is translation initiated in the 70S subunit of prokaryotic ribosomes?
The 50S subunit joins the 30S initiation complex to form the 70S initiation complex
Initiation factors are released and GTP is hydrolysed
What happens in elongation?
Codon-directed binding of the incoming aminoacyl-tRNA
Peptide bond formation
Translocation (movement) of the ribosome along the mRNA in a 5’ → 3’ direction by the length of one codon (move 3 bases)
How is a peptide bond formed by peptidyl transferase?
The aminoacyl portion of fMet-tRNA is transferred to the amino acid group of
the amino acid (aa) residue in the A site, forming a peptide bond
Activity due to the ribozyme function of 23S
Overall Keq ~1. therefore no energy input is required
Overview o the mechanism of translocation
Elongation factors attached to incoming amino acid (aa) tRNA moves into vacant A site
Proof reading step to make sure right aa is attached to the right tRNA
GTP hydrolysis allows for reorganisation of tRNA, allowing it to come into contact with the tRNA in the P site, allowing peptide transferase to occur
Peptide chain transfers to A site tRNA
EF-G facilitates translocation of ribosome along the mRNA
EF-G slots into partial A site, hydrolysis of GTP forces the EF-G into the rest of the A site and pulls the ribosome along.
Peptide now in the P site, A site now empty again, previous tRNA now in exit site
What is the role of the elongation factors EF-Tu and EF-G?
EF-Tu brings in each aa-tRNA
EF-G facilitates translocation to next codon
What does the binding of the incoming aminoacyl-tRNA require?
A soluble supernatant factor, elongation factor T (EF-T) and GTP
What are the two polypeptides of EF-T?
EF-Tu, 45kDa (heat unstable)
EF=Ts, 30kDa (heat stale)
What are the characteristics of EF-Tu?
EF-Tu is very abundant (~20 mols per ribosome). Most aa-tRNAs in the cell are complexed with EF-Tu
EF-Tu does not react with fmet-tRNAmet explaining why this is not bound during elongation
When bound to EF-Tu the labile ester bond between the tRNA and aminoacyl residue is protected from hydrolysis
How does EF-Tu interact with the Active (A) site of the ribosome?
Bends the A site to hold active site away until proofreading has happened and check the right aa, tRNA and the right codon are all matching
EF-Tu released from A by GTP hydrolysis, A site returns to original position
What is EF-Tu proofreading role in translation?
It takes a few milliseconds for GTP hydrolysis to occur, and a few more milliseconds for EF-Tu-GDP release. Only after EF-TU-GDP release can peptide bond formation occur. These intervals provide the opportunity for a weakly bound, non-cognate aa-tRNA (incorrect codon-anticodon match) to dissociate from the ribosome
What is the movement of the ribosome along the mRNA in translocation effected by?
Elongation factor G/GTP (EF-G/GTP) in bacteria or EF-2 in eukaryotes
What is EF-G structurally similar to?
The structure of EF-G is very similar to the EF-Tu
The structure of the N-terminal region of EF-G mimics the tRNA