Drop-off-reinitiation at the N-termini of nascent peptides and its regulation by IF3, EF-G, and RRF.

In translation initiation in prokaryotes, IF3 recognizes the interaction between initiator codon of mRNA and anticodon of fMet-tRNA ini and then relocates the fMet-tRNA ini to an active position. Here we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, e.g. Watson-Crick, are more preferred, but other types of base pairs, e.g. G/U wobble, are also tolerated; Weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNA ini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the N-terminal fMet. We refer to this event as the N-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNA ini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the N-terminal drop-off-reinitiation.