Non-canonical Escherichia coli transcripts lacking a Shine-Dalgarno motif have very different translational efficiencies and do not form a coherent group.

Translation initiation in 50-70 % of transcripts in Escherichia coli requires base pairing between the Shine-Dalgarno (SD) motif in the mRNA and the anti-SD motif at the 3' end of the 16S rRNA. However, 30-50 % of E. coli transcripts are non-canonical and are not preceded by an SD motif. The 5' ends of 44 E. coli transcripts were determined, all of which contained a 5'-UTR (no leaderless transcripts), but only a minority contained an SD motif. The 5'-UTR lengths were compared with those listed in RegulonDB and reported in previous publications, and the identities and differences were obtained in all possible combinations. We aimed to quantify the translational efficiencies of non-canonical 5'-UTRs using GusA reporter gene assays and Northern blot analyses. Ten non-canonical 5'-UTRs and two control 5'-UTRs with an SD motif were cloned upstream of the gusA gene. The translational efficiencies were quantified under five different conditions (different growth rates via two different temperatures and two different carbon sources, and heat shock). The translational efficiencies of the non-canonical 5'-UTRs varied widely, from 5 to 384 % of the positive control. In addition, the non-canonical transcripts did not exhibit a common regulatory pattern with changing environmental parameters. No correlation could be observed between the translational efficiencies of the non-canonical 5'-UTRs and their lengths, sequences, GC content, or predicted secondary structures. The introduction of an SD motif enhanced the translational efficiency of a poorly translated non-canonical transcript, while the efficiency of a well-translated non-canonical transcript remained unchanged. Taken together, the mechanisms of translation initiation at non-canonical transcripts in E. coli still need to be elucidated.