Initiating translation in mycobacteria: ribosome complexity in a leaderless bacterium

SUMMARYDecades of research have led to a detailed mechanistic understanding of bacterial translation initiation. Almostall this work has focused on ?canonical? translation initiation, which involves association of the 16S ribosomalRNA with the mRNA at a Shine-Dalgarno (SD) sequence. Our studies, along with very recent publications,have uncovered a surprisingly large class of non-canonical bacterial mRNAs: leaderless mRNAs (LLmRNAs)lack a 5?-untranslated region (UTR) and the critical SD sequence. LLmRNAs are rare in E. coli and havereceived little attention. However, our published and preliminary data indicate that leaderless translationinitiation is both common and robust in mycobacteria (~25% of all mRNAs). The surprising discovery thatleaderless-pervasive bacteria are far more common than once thought identifies a critical gap in ourunderstanding of ribosome biology and translation in bacteria and, therefore, is of fundamental biologicalsignificance. Ribosomes are major antibiotic targets, but leaderless translation represents a new essentialprocess for which novel inhibitors could be identified and developed as anti-mycobacterial agents.The goal of this proposal is to determine the mechanism by which ribosomes recognize, bind and initiatetranslation on an LLmRNA in the absence of a 5?UTR. Our preliminary data suggest that leaderless translationis achieved by an initiation complex that includes a 70S ribosome bound to mycobacterial RNA polymerase atthe site of transcription initiation, suggesting that nascent mRNA feeds directly into the ribosome, obviating theneed for a SD sequence. While co-transcription and translation is an established paradigm for bacteria, it hasonly been considered in the context of the elongating complex on a leadered mRNA. Our data support a simplemodel of co-initiation of transcription and translation that allows efficient translation of LLmRNAs found inbacteria, archaea and mitochondria. In the proposed work, we aim to identify and characterize initiationcomplexes that enable leaderless translation in mycobacteria. M. smegmatis is ideal for the proposed workbecause it is genetically tractable, which will facilitate the first mechanistic study of LLmRNA translation in abacterial species that natively expresses large numbers of leaderless transcripts. Our collective expertise inmycobacterial ribosome structure and biology has provided a solid foundation for the proposed studies. Wehave developed or are developing complementary in vitro and in vivo tools and assays that are now set tofocus on dissecting leaderless initiation complexes, to catch up with the well-described models of canonicalleadered translation initiation. Our potent proposed combination of traditional in vitro biochemistry with newinitiation complex inhibitors and in vivo ribosome profiling will promote a deeper understanding of the complexprocess of leaderless gene expression in mycobacteria.