RNA-Protein Interactions in Bacterial RNase P

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0091081 Venkat GopalanThe long term goal of this research is to understand RNA-protein interactions in Escherichia coli RNase P, an enzyme essential for the processing precursor tRNAs (ptRNAs) to their mature forms. E. coli RNase P, a ribonucleoprotein (RNP) complex, consists of a catalytic RNA subunit (M1 RNA) and a protein cofactor (C5 protein); both subunits are essential for its catalytic activity in vivo. The role of C5 protein in RNase P catalysis is distinct from other protein-facilitated RNA-catalyzed reactions in that it enhances the catalytic efficiency and versatility of a catalytic RNA which acts in trans on numerous substrates. Recent studies have demonstrated that the protein not only enhances the affinity of the substrate for the catalytic RNA subunit but also increases the rate of the RNA-catalyzed cleavage reaction. Several questions with regard to the mechanism of action of this unique catalytic RNP complex remain unanswered. In this project, a combination of biochemical and biophysical approaches will be used to determine the role of C5 protein in assembling a functional RNase P complex. The first objective of this study will be to employ structure-based mutagenesis and a genetic complementation assay to identify amino acid residues in C5 protein that are essential for its function in vivo. The second objective is to dissect the mechanisms by which the protein cofactor can exert effects on substrate recognition as well as catalysis. Lastly, by rational design of C5 mutants bearing cysteine residues at various positions in the protein molecule and modifying the cysteine residues with thiol-specific crosslinking, footprinting and spectroscopic probes, low resolution information regarding contact sites between (i) C5 protein and M1 RNA, and (ii) C5 protein and ptRNA substrates, will be obtained and used to gain structural perspectives critical for elucidating the mechanism of action of RNase P. RNA-protein interactions play an important role in numerous regulatory systems in vivo (e.g., translational control of gene expression). Results from this study will serve as a paradigm for understanding intermolecular interactions in other RNPs that control various prokaryotic and eukaryotic cellular processes. Most of the experiments described above are in progress and will rapidly furnish valuable insights. In addition to integrating laboratory research and education for several undergraduate and graduate students, this project has already helped renew and nurture several academic collaborations.