Genome-wide analysis of programmed ribosome stalling

Certain nascent chains can interact with the exit tunnel, triggering the ribosome to indefinitely pause along the transcript on which they are translating. Such examples of peptide induced pausing have been commonly found at regulatory open reading frames of polycistronic genes upstream of those encoding for functional proteins. In one specific case, the interaction between the nascent chain encoded by ermCL and the m2 methyl group of residue A2503 of the 23S rRNA facilitates in the expression of downstream gene ermC. Moreover, this interaction is further stabilized by erythromycin, which then induces structural rearrangement of the polycistronic message, thereby exposing an internal ribosome binding site required for the synthesis of ErmC. RlmN is the methyltransferase that is responsible for methylating A2503, serving as the posttranscriptional modification necessary for signaling translation arrest at ermCL. However, little is understood about the mechanism of peptide induced stalling on the ribosome. In order to address this we employed ribosome profiling, which entails deep sequencing ribosome-protected mRNA footprints, to comprehensively monitor in vivo translation rates on cells lacking rlmN to systematically identify other nascent chains that stall through m²A2503 and globally evaluate translational efficiency caused by the lack of this methylation. More generally, this study aims to examine the patterns of gene expression regulated through ribosome stalling and further elucidate mechanisms that govern interactions between the nascent chain and the ribosomal exit tunnel.