Predicted Bacterial uRBSs Reveal Translational Coupling and Ribosome-Mediated RBS Occlusion as Gene-Controlling Mechanisms

Abstract Upstream open reading frames (uORFs) in the 5′ leader of bacterial mRNAs can modulate gene expression, yet genome-wide identification remains limited. We combined bioinformatic prediction of ribosome-binding sites (RBSs)—a Shine-Dalgarno sequence and a start codon—with experimental validation to uncover new uORFs in Sinorhizobium meliloti 2011. From totally 1106 predicted upstream RBSs (uRBSs), we first examined 15 candidates using eGFP reporters and integrating existing RNA-seq and Ribo-seq data. Translation was detected at 13 sites, with fluorescence intensity broadly correlating with predicted initiation rates. Two uRBSs correspond to gene start sites, thereby refining gene annotations. In nine cases, uRBS mutations affected downstream gene expression in reporter fusions. Among others, the data suggests that a Type I secretion system operon, the RNA chaperone gene hfq, and metabolic genes are regulated by uORFs. Four uORFs acted through translational coupling. We also identified uRBSs that were ribosome-occupied yet (nearly) silent in eGFP assays, and closely spaced to the downstream main RBS (mRBS). These uRBSs probably mediate ribosomal occlusion downregulating lacR and SM2011_RS36230. A re-screen of the prediction set revealed 335 close uRBS/mRBS pairs. Three of them were analyzed, supporting the proposed ribosomal occlusion mechanism for SM2011_RS03630 and SM2011_RS22110, while for glnK translational coupling to an uORF was suggested. These results indicate that uORFs are more widespread in bacteria than previously recognized and suggest that direct ribosomal occlusion of the mRBS is a novel mechanism for down-regulating protein synthesis.