Mechanism of Translation

The ribosome is the cell's protein-synthesising factory, and the mechanism by which it carries out translation unfolds in the same three phases, initiation, elongation and termination, that also structure transcription, though the molecular machinery involved is entirely different. Before translation of a given mRNA can begin, the reading frame -- one of several possible ways of dividing a sequence of bases into successive triplet codons -- has to be fixed by locating the correct start codon; any stretch of RNA that begins with a start codon and runs, uninterrupted, through to a stop codon, and which can therefore actually be translated into a protein, is called an open reading frame (ORF), and a complete mRNA translational unit is precisely the stretch of RNA bounded by that start codon and that stop codon. Beyond the coding sequence itself, mRNA carries additional stretches at both ends that are never translated, called untranslated regions (UTRs): one before the start codon at the 5' end, and one after the stop codon at the 3' end. Translation always starts at an AUG codon, which is specifically recognised by a dedicated initiator tRNA charged with the amino acid methionine (in prokaryotes this initiator carries a chemically modified form, N-formylmethionine, or fMet, while eukaryotes use ordinary, unmodified methionine at this position). In bacteria, the free 5' end of the mRNA carries a special short sequence just upstream of the start codon, called the Shine-Dalgarno (S-D) sequence, that base-pairs directly with a complementary region of the small subunit's 16S rRNA and thereby helps position the ribosome correctly at the true start codon. Initiation itself, in E. coli, begins with assembly of an initiation complex from the free 30S ribosomal subunit, the mRNA, the charged initiator tRNA (fMet-tRNA-fMet), three dedicated initiation factor proteins (IF1, IF2 and IF3), GTP and Mg2+ ions: IF3 first binds the 30S subunit and enables it to engage the mRNA, after which IF2 promotes binding of the charged initiator tRNA to the small subunit specifically at the AUG codon, an event that fixes the reading frame for every codon that follows. Once this assembly is complete, IF3 is released and the large 50S subunit joins to form the complete 70S ribosome, consuming one molecule of GTP in the process and releasing the remaining initiation factors. Elongation then proceeds as a repeating cycle at three functionally distinct sites within the assembled ribosome, the aminoacyl (A) site, the peptidyl (P) site and the exit (E) site: the charged initiator tRNA occupies the P site to begin with, a second charged tRNA is delivered into the vacant A site (with the help of elongation factors EF-Tu and EF-Ts, plus GTP) and pairs its anticodon with the second codon on the mRNA, the enzyme peptidyl transferase then forms a peptide bond joining the two amino acids while simultaneously breaking the bond linking the first amino acid to its now-spent tRNA, and the entire mRNA-tRNA-peptide assembly is then translocated, at the cost of further GTP hydrolysis, by exactly one codon (three nucleotides) toward the P site, shifting the spent tRNA into the E site for release and freeing the A site to accept the next charged tRNA; this whole cycle then simply repeats, adding one further amino acid to the growing chain with each turn. Translation finally terminates once one of the three stop codons (UAA, UAG or UGA) enters the A site; because no tRNA exists that recognises a stop codon, a GTP-dependent release factor binds there instead, triggering release of the completed polypeptide chain from the final tRNA, after which that tRNA is itself released and the ribosome dissociates back into its separate large and s …