Evidence for the pairing of mRNA exiting ribosomes acting as a Driver of unidirectional forward bypassing: Its cessation permits Backwards scanning

Abstract

Efficient translational avoidance of a 50 nucleotide non-coding insert in phage T4 gene 60 sequence involves dissociation of peptidyl-tRNA pairing from the mRNA take-off codon, GGA. This tRNA is retained in the ribosomal P-site during bypassing. Its anticodon does not scan the 5- prime part of the non-coding sequence, which contains a cognate GGG codon, but it does scan the main and 3-prime part of the coding gap for potential complementarity. Re-pairing to a matched GGA landing site codon occurs exclusively 48-50 nt after take-off. Coding resumption ensues. Here we analyze proteins synthesized from mutants that abrogate the potential for re-pairing at the canonical landing site. The results show that some ribosomes move backwards 39 nt allowing re-pairing to the initially hidden GGG or forwards at least 56 additional nt before resumed anticodon: codon pairing at a downstream matched codon. We discuss the nature of the driving force for the initial unidirectional movement and why it becomes dissipated when the site of landing in WT is reached. Also presented is an analysis of the relative importance of recoding signals located 6 nucleotides upstream (5-prime) and close downstream (3-prime) of the WT landing site, for re-pairing to mRNA and coding resumption.