Lecture 5

1. INITIATION

Assembly of active ribosome by placing the first mRNA codon (AUG or START codon) near the P site and pairing it with initiation tRNA, fMet-tRNA^fMet.
Occurs once per polypeptide chain or molecule.

2. ELONGATION

Involves three distinct steps for each mRNA codon or amino acid in the new polypeptide chain:

1. Transfer of proper aminoacyl-tRNA from cytoplasm to A-site of ribosome

2. Covalent linkage of new amino acid to growing polypeptide chain - peptidyl transfer.

3. Movement of tRNA from A-site to P-site and simultaneous movement of mRNA by 3 nucleotides - translocation.

Occurs multiple times per polypeptide.

3. TERMINATION

Reading of final mRNA codon (STOP codon) and dissociation of polypeptide from ribosome.
Occurs once per polypeptide chain.

ELONGATION OF PROTEIN SYNTHESIS

3 distinct steps to add one amino acid to the growing polypeptide chain.
Occurs many times per polypeptide, the number of which depends upon the number of mRNA codons or amino acids in the protein
The Elongation Cycle is similar in prokaryotes and eukaryotes.
Fast: 15-20 amino acids added per second
Accurate: 1 mistake every ~10,000 amino acids

Transfer of Aminoacyl-tRNA

Non-initiator, aminoacyl-tRNA is placed in the ribosomal A-site over the mRNA codon in such a way that base pairing occurs between the anticodon loop of the tRNA and mRNA codon (see lecture 1).

Aminoacyl-tRNA transfer is facilitated by two soluble protein transfer factors, called elongation factors, EF-Tu and EF-Ts in prokaryotes:

EF-Tu binds GDP and GTP and is a model G protein.
EF-Ts exchanges GDP for GTP on EF-Tu.

The elongation factors are similar in eukaryotes. Instead of two proteins, there is a stable trimer, eEF1-alpha-beta-gamma, which carries out the same function as EF-Tu and EF-Ts. eEF1-alpha is the eukaryotic equivalent of EF-Tu, and eEF1-beta-gamma the eukaryotic equivalent of EF-Ts.

EF-Tu-GDP is the inactive form. EF-Ts activates EF-Tu by catalyzing the exchange of GDP for GTP. EF-Tu-GTP is the active form which binds to non-initiator tRNAs to which the aminoacyl group has been attached.

EF-Tu-GTP-aminoacyl-tRNA is then carried to the ribosome. The complex binds to the ribosome, with the aminoacyl-tRNA in the A-site. Ribosome binding stimulates GTP hydrolysis and EF-Tu-GDP dissociates from the ribosome, free to recycle through the step multiple times.

EF-Tu is partially responsible for the high degree of accuracy of protein synthesis via a mechanism called kinetic proofreading. The error rate of protein synthesis is only 1 wrong amino acid for every 10,000 amino acids added to polypeptides.

When a charged tRNA is positioned into the A-site, the anticodon must base pair with the mRNA codon. If there is an incorrect match, the incorrect aa-tRNA dissociates from the ribosome before GTP hydrolysis occurs. If there is a correct match, GTP hydrolysis occurs and EF-Tu-GDP leaves the ribosome before the cognate aminoacyl-tRNA can dissociate and EF-Tu-GDP dissociates instead, leaving the correct tRNA on the ribosome.

EF-Tu is so important to cellular function that it is one of the most abundant cytoplasmic proteins (>5%). There is one copy of the EF-Tu protein for each tRNA molecule in the cell.

Next: Peptidyl Transfer

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