CAREER: Mechanisms of RNA-mediated control of gene expression in bacteria

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Title: CAREER: Mechanisms of RNA-mediated control of gene expression in bacteriaBacteria have evolved multiple means to sense their environment and respond to changing conditions, including temperature. One strategy that some bacteria have evolved to sense temperature is the placement of temperature-responsive sequences in messenger RNAs. These ‘RNA thermosensors’ change their structure in response to changing temperature, exerting control over the production of particular proteins. This project will investigate the structural requirements for temperature sensitivity and elucidate RNA-RNA interactions that impact bacterial virulence. This research will report on ubiquitous and important process in RNA-mediated mechanisms including melting and annealing. This work will inform on RNA molecular recognition and the RNA structure-function relationship. Further insights into RNA structure, stability, and intermolecular interactions will aid in the engineering of new RNA-based biosensors. This project will also integrate research and education to foster scientific training and education of high school, undergraduate, and graduate students. As a component of this project, undergraduate students will be trained in cutting-edge biomolecular nuclear magnetic resonance (NMR) spectroscopy and will contribute to a public database containing RNA chemical shift information. Outreach activities aimed at engaging young students from traditionally underrepresented backgrounds will include demonstration of magnetic resonance imaging techniques. The overarching scientific objective of this project is to gain insight into how non-coding RNA molecules from the bacterial pathogen L. monocytogenes function independently and in concert to control gene expression. An RNA thermosensor controls the translation of a key transcriptional activator, PrfA, which ultimately controls the expression of a cluster of virulence genes. Objective 1 will reveal the biochemical and biophysical properties that govern temperature sensing in RNA thermosensors. Objective 2 will characterize, in atomic detail, the structure and conformational changes that occur in the prfA RNA thermosensor at various temperatures. Objective 3 will elucidate the structural and molecular link between the RNA thermosensor and a metabolite-binding riboswitch, which function together to mediate expression of virulence genes. To accomplish these objectives, we will use a multidisciplinary approach combining biochemical, biophysics, and structural techniques including NMR spectroscopy, small angle X-ray scattering, and X-ray crystallography. This project will lead to a deeper understanding of how certain pathogenic bacteria sense and respond to temperature signals and how RNA molecules remodel and interact to control gene expression. This project is supported by the Molecular Biophysics and Genetic Mechanism clusters of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.