Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5' terminus of its mRNA. The 5'-terminal AUG (5'-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5'-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5'-uAUG. Additional sequences within the ptrB 5' untranslated region (5'-UTR) work cumulatively with the 5'-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5'-UTRs from other mRNAs with the ptrB 5'-UTR sequence showed a similar dependence on the 5'-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5'-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5'-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5' termini of E. coli mRNAs and the ability of ptrB 5'-UTR regulation to function independently of gene context, the regulatory effects of 5'-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.