| RNase P is the ubiquitous and universally conserved RNA enzyme that catalyzes a phosphodiester cleavage to remove the 5' precursor sequence of all tRNAs. In bacteria, the small RNase P protein (∼14 kDa) is required to activate RNase P RNA (∼130 kDa) in vivo, but previous studies have yielded contradictory conclusions regarding its specific function. This thesis is aimed towards elucidating the function of the bacterial RNase P protein and determining the structural and mechanistic properties required for RNase P RNA activation. Biochemical and biophysical techniques are used to examine previously proposed functions of the protein in holoenzyme and enzyme-substrate assembly. The results demonstrate that the protein can influence the global structural stability of the RNA, dimer formation by the holoenzyme and precursor tRNA recognition, but it has different effects in Escherichia coli and Bacillus subtilis holoenzymes, the main experimental models. Comparison of E. coli and B. subtilis RNase P proteins in complex with both their cognate and noncognate RNAs demonstrates that differences between the two holoenzymes reside in the RNA and not the protein components of each. The results support a model where the protein activates the intrinsic RNA properties necessary for substrate binding.; In order to examine the specific effects of the protein on substrate binding and catalysis, a series of pre-steady state and steady state kinetic experiments were conducted. The results demonstrate that the protein specifically increases the association rate of the substrate and does not have a substantial effect on any other rate constant. To gain a structural perspective on this protein effect, phosphorothiate-I2 and dimethylsulfate structure-mapping experiments were conducted on the RNA in the absence and presence of protein, with a parallel examination of the tRNA binding site. These data mapped onto the crystal structure of Bacillus stearothermophilus RNase P RNA (recently solved at 3.3 A resolution) support a model whereby the protein binds to a conserved region of the RNA and influences the adjacent RNA structure that contacts substrate. |
