Lecture 8 Bio 99

Elongation steps

1. Aminoacyl-tRNA enters ribosome (Ef-Tu)

2. Peptide bond forms

3. Ribosome shifts for (Ef-G)

Ef-T recharges Ef-Tu, replacing its GDP with GTP

Elongation Step 1

• Ef-tu escorts new aminoacyl-tRNA into A site.

• GTP hydrolyzed to GDP, and Ef-tu leaves, bound to the GDP.

• Ef-T recharges, switching the GDP for a new GTP.

• Accomodation: correct tRNA base-pairing brings its amino acid close to polypeptide.

Elongation Step 2

Peptidyl Transferase Rxn

Catalyzed by the large subunit (Magnesium). The amino group of aa in the A site attacks the carbonyl of the peptide in the P-site tRNA, forming a new peptide bond and transferring the growing chain to the A-site tRNA.

What results from the peptidyl transferase rxn?

• Peptide bond forms

• Aminoacyl-tRNA bond of P site is severed, so A site’s tRNA is where the

  growing polypeptide is now linked

Creates hybrid state, where the tRNAs are caught between their old and new positions.

Elongation Step 3

Translocation

• Solves the hybrid state, ribosome needs to shift over.

• Driven by Ef-G and GTP hydrolysis.

• Ef-G enters A site, the hydrolysis pulls the ribosome over one codon.

• Ef-G : GDP released

Termination Step 1

Ribosome finds a STOP codon.

• There is no tRNA that recognizes a stop codon, RF factor does.

• RF mimics shape of tRNA, drives last peptidyl rxn.

• Polypeptide releases.

Termination Step 2

RF Removal

• RF-3 kicks out RF using GTP hydrolysis

What bacterial proteins mimic tRNAs?

Rfs and Efs, allows them to enter A site

Which bacterial proteins are involved in GTP or ATP binding or

hydrolysis?

ATP

• AATS

GTP

• RF3

• IF-2

• EF-Tu

• EF-G

Puromycin

Puromycin is an aminoacyl-tRNA mimic that enters the A site of the ribosome, accepts the growing polypeptide chain, but nothing can be attached to it, terminating translation.

Non-stop mRNA Decay

When there’s no stop codon, translation continues

• Translates poly-A tail to poly-lysine recognized by Ski 7

• Ski 7 dissociates ribosome, exosome eats mRNA, recruits protease to eat protein.

Nonsense-mediated mRNA Decay

Premature Stop Codon

• EJCs aren’t being displaced like usual.

• Upf1 and Upf2 recognize EJCs, recruit enzyme to decap 5’ of mRNA

• Causes rapid degradation