Initiation at AUGUG and GUGUG sequences can lead to translation of overlapping reading frames in E. coli.

During initiation, the ribosome is tasked to efficiently recognize open reading frames (ORFs) for accurate and fast translation of mRNAs. A critical step is start codon recognition, which is modulated by initiation factors, mRNA structure, a Shine Dalgarno (SD) sequence and the start codon itself. Within the Escherichia coli genome, we identified more than 50 annotated initiation sites harboring AUGUG or GUGUG sequence motifs that provide two canonical start codons, AUG and GUG, in immediate proximity. As these sites may challenge start codon recognition, we studied if and how the ribosome is accurately guided to the designated ORF, with a special focus on the SD sequence as well as adenine at the fourth coding sequence position (A4). By in vitro and in vivo experiments, we characterized key requirements for unambiguous start codon recognition, but also discovered initiation sites that lead to the translation of both overlapping reading frames. Our findings corroborate the existence of an ambiguous translation initiation mechanism, implicating a multitude of so far unrecognized ORFs and translation products in bacteria.

Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus.

Noncoding RNAs, including regulatory RNAs (sRNAs), are instrumental in regulating gene expression in pathogenic bacteria, allowing them to adapt to various stresses encountered in their host environments. Staphylococcus aureus is a well-studied model for RNA-mediated regulation of virulence and pathogenicity, with sRNAs playing significant roles in shaping S. aureus interactions with human and animal hosts. By modulating the translation and/or stability of target mRNAs, sRNAs regulate the synthesis of virulence factors and regulatory proteins required for pathogenesis. Moreover, perturbation of the levels of RNA modifications in two other classes of noncoding RNAs, rRNAs, and tRNAs, has been proposed to contribute to stress adaptation. However, the study of how these various factors affect translation regulation has often been restricted to specific genes, using in vivo reporters and/or in vitro translation systems. Genome-wide sequencing approaches offer novel perspectives for studying RNA-dependent regulation. In particular, ribosome profiling methods provide a powerful resource for characterizing the overall landscape of translational regulation, contributing to a better understanding of S. aureus physiopathology. Here, we describe protocols that we have adapted to perform ribosome profiling in S. aureus.