RNA Structure and Dynamics In Vivo

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Intellectual Merit. The overarching vision of this research is to create a technology for analyzing RNA structure at single nucleotide resolution for RNAs of arbitrary complexity inside living cells. The goal is timely because of the growing prominence of RNA in all fields of biology. This prominence reflects, in part, the appreciation that the ribosome and many other ribonucleoproteins are highly dynamic and nimble biomolecular machines. It also reflects the many new roles being discovered for the numerous untranslated and regulatory RNAs with critical functions. The goal of this research program is therefore to extend the high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) RNA structure analysis technology, invented in the project laboratory, into a robust tool for in vivo analysis of RNA structure. This technology will then be used to understand surprising discoveries that preliminary studies have revealed about RNA structure in E. coli ribosomes as they exist inside healthy, growing, bacterial cells. SHAPE technology is also sufficiently mature to serve as the basis for a completely undergraduate-driven research endeavor, which will be developed into the Undergraduate Transcriptome Project (UTP).Broader Impacts are envisioned to be multi-fold. An accurate and efficient technology for analyzing RNA structure in vivo has the potential to transform many fields in the biological sciences. When fully developed under this research program, in vivo SHAPE will interconnect molecular information about RNA structure as obtained from high-resolution structural and biochemical approaches in highly purified systems with cellular function and fitness in the complex in vivo environment. The technology created in this work will be disseminated widely via both formal and informal channels. The Undergraduate Transcriptome Project (UTP) is a new kind of undergraduate-driven research experience. Students will be mentored to assume a large part of the responsibility for the project and will initiate, lead, and maintain its intellectual momentum. The UTP will focus on structural analysis of the RNA genome of an icosahedral plant virus. Such viruses are among the most widely distributed plant, animal and agricultural pathogens in biology. The project has several distinctive features with the goal of inspiringundergraduates to become deeply invested in and passionate about scientific research.