This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In all kingdoms of life RNAs are modified both during and after synthesis, thereby altering the stability, higher-order structure, and activity of RNA through changes in base stacking and hydrogen bonding. These generally conserved modifications are located at key functional regions and are required for optimal RNA function. Approximately 0.8% of the coding capacity of the E.coli cell is dedicated to enzymes that modify RNA with unique or limited multisite specificity.
The aim of this proposal is to determine the basis for selectivity and catalysis in two of the most abundant families of RNA modifying enzymes: 5-methyl uracil (m5U0 methyltransferases (MTases) and pseudouridine synthases (PS). Our reported structures of two m5U MTases have yelded a model for specific recognition in which the RNA substrate is refolded onto the enzyme and a base is flipped out into the active site. This work proposes to test, refine and elaborate that model in kinetic and structural terms. Our to-date and proposed studies of five sub-classes of bacterial PS that modify stem loops of tRNAs (TruA and TruB), a stem loop (RluD), and a lehix (RluB and RluF) of rRNA is yielding models for site specificity and regional specificity that will serve as paradigms for PS that play roles in human health. The field will thus be advanced into regulation in human biology with our proposed work on human Pus1 and Pus3, two enzymes that regulate nuclear receptor signaling by pseudouridylating an RNA activator of nuclear receptors (steroid receptor RNA activator, SRA). Defects in thse enzymes cause sideroblastic anemia. We seek to determine the structure of a human tRNA adenine MTase as a potential new anti-HIV drug target. It generates an essential modification that controls reverse transcription of HIV RNA. Mass Spectroscopy analysis will facilitate the identification of purified proteins and eventually the identification of post-translational modification.
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