9631039 Nolan The focus of the proposed research program is a detailed structural analysis of the bacterial ribonuclease P (RNase P) holoenzyme. Bacterial RNase P cleaves 5'-precursor sequences from tRNA transcripts. It consists of a large (140 kd) RNA and a small (14 kd) protein subunit. In vitro the RNA subunit is the catalytic moiety and the protein is dispensable. However, the RNase P protein is essential for viability in vivo, and the kinetics of the holoenzyme reaction in vitro differs dramatically from the RNA-alone reaction in salt requirement, substrate specificity, and reaction rate. While the catalytic RNA subunit of the enzyme is well characterized, little is known about how the protein moiety mediates its pleiotropic effects on the enzymatic reaction. Specific problems addressed are: (a) Localization of the protein binding site on RNase P RNA, and (b) Identification of protein domains required for function. These problems will be studied using chemical and enzymatic footprinting, crosslink analysis, mutational analysis, and kinetic studies. The proposed studies will shed light on the RNase P protein and its function in the holoenzyme; this will enhance understanding of the RNA enzyme mechanism, and RNA-protein as well as RNA-RNA interactions. %%% The focus of the proposed research program is a detailed structural analysis of the bacterial ribonuclease P (RNase P) holoenzyme. A great deal is known about the catalytic RNA subunit of the enzyme, but little is known about how the protein moiety mediates its pleiotropic effects on the enzymatic reaction, which are required for function in vivo. The proposed experiments will contribute to the understanding of how the protein subunit interacts with the RNA and how this interaction affects the reaction mechanism. ***
