Visualizing Shine-Dalgarno independent translation initiation in bacteria by cryo-electron microscopy

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In all cells, ribosomes translate the genetic code, synthesizing proteins based on the information content of mRNA. The rate of translation varies dramatically, depending on the sequence of the translation initiation region (TIR) of the mRNA, which lies upstream from the start codon. In bacteria, a well-known feature of the TIR is the Shine-Dalgarno (SD) sequence, a purine-rich element located 7-12 nucleotides upstream from the start codon. The SD pairs with the anti-Shine-Dalgarno (ASD), a pyrimidine-rich element at the 3' end of the 16S rRNA (a component of the 30S ribosomal subunit). SD-ASD pairing facilitates start codon recognition and promotes initiation of the protein translation process. As the ASD of 16S rRNA is universally conserved, it has long been presumed that SD-ASD pairing contributes to start codon recognition in all bacteria. However, recent genome sequencing studies have revealed that whole taxonomic groups of bacteria lack SD sequences (and yet retain the conserved ASD). These include the phyla Bacteroidetes. This raises questions: (i) Is there a mechanism that prevents the ASD from pairing to mRNA during initiation? (ii) What are the initiation mechanisms in these organisms? The long-term goal of our research program is to understand the biology of the ribosome. In the research proposed here, we aim to address these questions in the Bacteroidetes, using Flavobacterium johnsoniae as the model organism. The specific objectives are: (1) Determine the structural determinants preventing SD-ASD interaction in the Bacteroidetes initiation complex. (2) Elucidating the role of initiation factors in SD-independent translation initiation. How translation initiation occurs in organism without the wide-spread SD-ASD interaction is an intrinsically interesting biological question. This taxonomic group of Bacteroidetes is large and includes important players of the gut microbiota of mammals. Yet the mechanisms of gene expression remain ill defined. The proposed studies employ a powerful set of complementary approaches, including cryo-electron microscopy to address fundamental questions of translation initiation in Bacteroidetes and may advance the field in a major way. Translation initiation appears to be naturally simplified in the Bacteroidetes, as the complication of SD-ASD pairing is effectively removed. Hence the proposed studies of F. johnsoniae initiation may clarify the roles of other players in translation initiation, such as the secondary structure of the TIR in the mRNA or the function of the ribosomal protein bS1. Such findings would hold broad relevance for all domains of life. The proposed research will also have broader impact. It will provide integrated laboratory research and training programs for graduate and postdoctoral students, including members of underrepresented minorities. The HQP from my laboratory will obtain a unique skill set that will make them competitive in biomedical and pharmaceutical research in Canada.