Operon mRNAs are organized into ORF-centric structures that predict translation efficiency

Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggests that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF. mRNA-seq and ribosome profiling of E. coli MG1655 (wild-type) cells at 37°C and DMS-seq of in vivo modified mRNA from wild-type cells, kasugamycin-treated or untreated ΔgcvB cells, as well as in vitro modified mRNA refolded at 37°C or denatured at 95°C. Reads from replicates of the same experiment were pooled in wiggle files.