Question

Provide detailed descriptions of the events of gene translation. Use E. coli as your model and include i.) initiation, ii.) elongation, and iii.) termination.

Answer 1

Translation is the process of making proteins from mRNA in ribosome.

It occurs in 3 steps : initiation, elongation and termination.

Initiation :

Activation of amino acid.  During the first stage of protein synthesis, taking place in the cytosol, aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each enzyme is specific for one amino acid and one or more corresponding tRNAs.

the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The mRNA then binds to the 30S subunit. The initiating (5')AUG is guided to its correct position by the Shine-Dalgarno sequence in the mRNA.

Bacterial ribosomes have three sites that bind tRNAs, the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site. The A and P sites bind to aminoacyl-tRNAs, whereas the E site binds only to uncharged tRNAs that have completed their task on the ribosome.

The initiating 5'AUG is positioned at the P site, the only site to which fMet-tRNAfMet can bind. The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site; during the subsequent elongation stage, all other incoming aminoacyl-tRNAs bind first to the A site. IF-1 binds at the A site and prevents tRNA binding at this site during initiation.

The initiation process , the complex consisting of the 30S ribosomal subunit, IF-3, and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNAfMet. The anticodon of this tRNA now pairs correctly with the mRNA’s initiation codon.  Now, this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi
, which are released from the complex. All three initiation factors depart from the ribosome at this point.

Elongation :

Elongation requires the initiation complex described above, aminoacyl-tRNAs, a set of three soluble cytosolic proteins called elongation factors (EF-Tu, EF-Ts, and EF-G), and 4 GTP.

In the first step of the elongation cycle, the
appropriate incoming aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu. The resulting aminoacyl-tRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex. The GTP is hydrolyzed and an EF-Tu–GDP complex is released from the 70S ribosome. The EF-Tu–GTP complex is regenerated in a process involving EF-Ts and GTP.

A peptide bond is now formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosome. This occurs by the transfer of the initiating N-formylmethionyl group from its tRNA to the amino group of the second amino acid, now in the A site .The -amino
group of the amino acid in the A site acts as a nucleophile, displacing the tRNA in the P site to form the peptide bond.

The enzymatic activity that catalyzes peptide bond formation has been referred to as peptidyl transferase.

In the final step of the elongation cycle, translocation, the ribosome moves one codon toward the 39 end of the mRNA. Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the energy provided by hydrolysis of another molecule of GTP. After translocation, the ribosome, with its attached dipeptidyl-tRNA and mRNA, is ready for the next elongation cycle and attachment of a third amino acid residue.

Termination :

Termination, the fourth stage of polypeptide synthesis, is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA), immediately following the final coded amino acid. Once a termination codon occupies the ribosomal A site, three termination factors, or release factors—the proteins RF-1, RF-2, and RF-3—contribute to (1) hydrolysis of
the terminal peptidyl-tRNA bond, (2) release of the free polypeptide and the last tRNA, now uncharged, from the P site, and (3) dissociation of the 70S ribosome into its 30S and 50S subunits, ready to start a new cycle of polypeptide synthesis. RF-1 recognizes the termination codons UAG and UAA, and RF-2 recognizes UGA and UAA. Either RF-1 or RF-2 (depending on which codon is present) binds at a termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid.