Dynamics of co-transcriptional riboswitch folding through single-molecule microscopy

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Project Abstract and Summary: RNA elements known as riboswitches are critical for proper genome expression and maintenance in many prokaryotes. Riboswitches are generally found upstream, in the 5’ untranslated region of an mRNA, and fold co-transcriptionally to structurally impede mRNA transcription and/or translation. In the presence of specific RNA binding ligands, riboswitches undergo alterations in secondary structure that either: 1) disallow translation initiation, or 2) result in altered transcription termination. Riboswitches are rarely found in eukaryotes, and thus present a novel vulnerability that can be targeted for anti-bacterial therapies. It has been clearly demonstrated in the literature that nascent RNA structure influences transcriptional activity of the transcribing polymerase in bulk assays, but little work has been done on how the transcribing polymerase itself influences nascent RNA structure in return. Further, RNA structure has been shown to impact active site conformations of the polymerase, but never in the context of the riboswitch. In this study we will comprehensively examine the mechanisms by which riboswitches influence the transcribing RNAP, and vice versa, using cutting edge single-molecule techniques. In addition to the novel science proposed in this application, this fellowship will further enhance my training, and strengthen me as an independent scientist. I will participate in seminars, workshops, mentoring and teach opportunities, as well as professional development and networking opportunities afforded to me by the diverse collection of programs offered at the University of Michigan (including programs organized by: the College of Literature, Sciences, and the Arts, the Life Sciences Institute, the Department of Chemistry, the Center for RNA Biomedicine, the Michigan Life Sciences Fellowship, the Office of Graduate Student and Postdoctoral Affairs, and the University of Michigan Postdoctoral Association). Further, Dr. Nils Walter has already demonstrated excellent mentorship skills, and his lab is comprised of a large, diverse group of postdoctoral fellows and graduate students with whom I can find support and guidance. Our lab also includes a large number of talented undergraduates which will provide me with many opportunities for collaborating with younger mentees. Overall, the research planned in this proposal, and the support granted to me by this program, will enrich both me and the field of riboswitch RNA biology.