Poster abstracts
Poster number 98 submitted by Courtney Niland
High-Throughput Enzymology Reveals Shared Molecular Recognition of Precursor tRNAs by the RNA and Protein Subunits of RNase P
Courtney N. Niland (Department of Biochemistry, Case Western Reserve University), Jing Zhao (Department of Biochemistry, Case Western Reserve University), Hsuan-Chun Lin (Department of Biochemistry, Case Western Reserve University), David R. Anderson (School of Business, CUNY Baruch College), Eckhard Jankowsky (Department of Biochemistry, Case Western Reserve University; Center for RNA Molecular Biology, Case Western Reserve University), Michael E. Harris (Department of Biochemistry, Case Western Reserve University)
Abstract:
The specificity of ribonucleoproteins is essential to their functions in biology. That function requires the ability to recognize cognate substrates from non-cognate binding sites in the cell. Ribonuclease P, RNase P, is a multi-substrate ribonucleoprotein enzyme that removes the 5’ leader from all pre-tRNAs despite variation in sequence and structure. Previous work demonstrated that the protein and RNA subunits of RNase P intimately contact nucleotides 5’ to the pre-tRNA cleavage site, the 5’ leader, but these positions vary considerably in the E. coli genome. The specificity of the enzyme for different 5’ leader sequences is poorly understood. E. coli RNase P was used to process pre-tRNA substrate pools randomized in their 5’ leader sequences in both the protein and RNA subunit binding sites (N(-1) to N(-6)). Using High-Throughput Sequencing Kinetics, HTS-Kin, to analyze this population we comprehensively determined the rate constants for processing of all possible 5’ leader sequences in the RNase P binding site. The resulting affinity distribution provides a complete description of enzyme specificity and reveals the full context dependence of mutations on processing rate. Importantly, these data sets reveal a strong influence of sequence identity in the binding site of the RNA subunit on sequence specificity and energetic contribution of contacts in the protein subunit binding site. Analyses of individual sequence variants confirmed the accuracy of the high throughput data and are being used to investigate the mechanistic basis for this strong energetic coupling between RNA-protein and RNA-RNA interactions. Surprisingly, this data also revealed pairing of the 5’ leader with the 3’ACCA as a positive determinant despite known enzyme contacts in this region. Further insight into the mechanistic basis for these effects will reveal how RNase P achieves specificity and provide deeper insight into molecular recognition by multi-substrate ribonucleoprotein enzymes.
Keywords: Enzymology, Ribonucleoprotein, RNase P