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

In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the 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 the codon, only Watson–Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson–Crick, are more preferred, but other types of base pairs, for example, 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 amino-terminal fMet. We refer to this event as the amino-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 amino-terminal drop-off-reinitiation.