Time-resolved hydroxyl radical footprinting of RNA

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DESCRIPTION (provided by applicant): The synthesis of new ribosomes is essential for cell growth, and tightly regulated in response to nutrients and stress. Defects in ribosome synthesis are linked to increased risk of cancer and a variety of congenital defects in rapidly growing tissues such as blood and skin. The aim of this research is to understand how the RNA and protein components of the ribosome assemble with each other to form the large and small subunits of the ribosome, and to understand how this process is accelerated and regulated by other proteins in the cell. Time-resolved footprinting can visualize changes in the RNA and protein interactions within 10-20 milliseconds, resulting in detailed information about the folding pathway of the RNA and RNA-protein recognition. This method will be used to investigate slow remodeling of RNA-protein complexes in the head of the 30S subunit, and study how maturation factors increase the rate of protein binding and assembly. A synchrotron X-ray beam will be used to footprint 30S assembly intermediates in living bacteria. The dynamics of a conformational switch in the rRNA will be observed directly using fluorescence spectroscopy of single RNA-protein complexes. The results of this research will increase understanding of how large RNA-protein complexes are necessary for cell growth, and contribute to a molecular explanation of human disorders arising from defects in the assembly of these complexes. PUBLIC HEALTH RELEVANCE: Ribosomes are a cellular complex that synthesizes new proteins, and cells must produce thousands of new ribosomes each minute to grow and divide. Deficiencies in ribosome production cause diseases of the blood, skin and other tissues, and have been linked to cancer. This research will visualize how the RNA and protein components of the ribosome come together, to learn how this process goes awry and how such defects may be corrected.