Structural studies of the RNA component of RNase P

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DESCRIPTION (provided by applicant): Transfer RNA is produced as a precursor molecule that needs to be processed both at its 3' and 5' ends. Ribonuclease RNase is the endonuclease for the 5' end of tRNA P, or P, only responsible processing by cleaving a precursor and leading to tRNA maturation. It contains an RNA and a protein component and has been identified in all organisms. It is one of the first catalytic RNA molecules identified and its study has been pivotal to our understanding of the role of RNA molecules in catalysis. RNase P is a true multi-turnover ribozyme and one of only two ribozymes conserved in all three kingdoms of life. The knowledge of the structure and function of RNase P promises to provide important and relevant information on a key ribozyme involved in a central cellular process common to all organisms and also to further our understanding of the structure and function of large RNA molecules. This proposal is concerned with the structure and function of bacterial RNase P. The RNA component of bacterial RNase P is made of two domains: a specificity and a catalytic domain. Recently, we solved the structure of the specificity domain of B. subtilis RNase P and we are now proposing to expand our studies to characterize more fully the specificity domain of B. subtilis RNase P, to study the structure of other bacterial RNase P, and to study the structure of the intact holoenzyme. The specific aims for this proposal are: 1) to determine the structure of the T. thermophilus RNase P specificity domain, 2) to obtain higher resolution information on the B. subtilis RNase P specificity domain, 3) to study the interactions of B. subtilis RNase P with metal ions and with its substrate, and 4) to crystallize and solve the structure of the intact B. subtilis RNase P holoenzyme. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the molecules that we require for our work, and X-ray crystallography to solve their structures.