Poster abstracts
Poster number 97 submitted by Courtney Niland
Investigating Shared Molecular Recognition By RNA and Protein Subunits of RNase P Using High-Throughput Sequencing Kinetics (HTS-Kin)
Courtney N. Niland (Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Jing Zhao (Department of Biochemistry, Case Western Reserve University, Cleveland, OH), David Anderson (School of Business, CUNY Baruch College), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Michael E. Harris (Department of Biochemistry, Case Western Reserve University, Cleveland, OH)
Abstract:
Ribonucleoproteins perform essential functions in biology that rely on their ability to recognize and process many different substrates. Currently an understanding of how the RNA and protein subunits of these enzymes work together to achieve substrate recognition and catalysis is lacking. Ribonuclease P, RNase P, a multi-substrate ribonucleoprotein, is made of a protein subunit and catalytic RNA subunit that removes the 5’ leader from all pre-tRNAs despite their variation in sequence and structure. Previous work from our lab and others indicate that both the protein and RNA subunits of RNase P make specific contacts in the 5’ leader of pre-tRNA. Using a new technique termed High-Throughput Sequencing Kinetics, HTS-Kin, we are able to analyze the effect of substrate variation in the 5’ leader on enzyme processing. This technique allows us to measure the processing rate constant for thousands of substrates in a single reaction and provides context dependence of mutations. The RNase P holoenzyme was placed into multiple turnover reactions with substrate pools randomized in the 5’ leader region contacting the protein subunit (-3 to -8) or the protein and RNA subunits (-1 to -6) and analyzed by HTS-Kin, providing a rate constant for nearly all 4096 substrates. The HTS-Kin results show a correlation between the identity of nucleotides contacting the RNA subunit and identity and contribution of those contacted by the protein subunit, indicative of thermodynamic coupling. Analyses of individual sequence variants is being used to confirm and investigate the basis for this strong thermodynamic coupling between RNA-protein and RNA-RNA interactions. HTS-Kin analysis of these same populations in single turnover reactions will reveal mechanistic detail and monitor the effect of nucleotides in the 5’ leader on catalysis. This information will reveal how RNase P achieves specificity and provide deeper insight into molecular recognition by multi-substrate ribonucleoprotein enzymes.
Keywords: RNase P, Ribonucleoprotein, Enzyme Kinetics