RNA -protein interactions in prokaryotic and eukaryotic ribonuclease P.

Ribonuclease P (RNase P) is the ubiquitous ribonucleoprotein enzyme responsible for cleaving all pre-tRNAs to form tRNA. In prokaryotes, RNase P has a single PRNA and a single P protein. In vitro, PRNA alone can catalyze the reaction under non-physiological conditions. In eukaryotes, RNase P has several proteins in addition to RNA; furthermore, all subunits are necessary for catalysis. Previous studies have revealed possible interaction sites between the PRNA and P protein. However, little is known about the nature of such intraholoenzyme interactions. This research contributes to the field by proving that the P protein and PRNA contains important ionic interactions in the prokaryotic RNase P holoenzyme. To measure the ionic strength dependence of the affinity between the B. subtilis PRNA and P protein, a magnetocapture-based assay was developed. At low ionic strength, increasing ionic strength enhances affinity due to stabilization of the PRNA structure. At high ionic strength, the affinity decreases as ionic strength increases, indicating that 3--4 ionic interactions stabilize holoenzyme formation. Point mutations implicate residues K5, R60, and R62 as potential ionic contacts between P protein and PRNA. The thermodynamic and kinetic effects of deletion mutants within PRNA demonstrate that important interactions with the P protein occur within the catalytic domain of PRNA. Taken together with chemical affinity cleavage experiments, the protein-RNA interface consists of helices P3, P4, and P19 of PRNA and the highly conserved RNR motif, N-terminus, and metal binding loop of P protein. Steady-state kinetic experiments demonstrate that multiple substrate binding sites occur in the S. cerevisiae holoenzyme. Unlike the prokaryotic enzyme, the yeast holoenzyme does not interact with the 5 ' leader. Instead, the characteristic 3' trailer of yeast tRNAs bind to at least one site of yeast RNase P. This site demonstrates a sequence preference for poly(U) and poly(G) since tRNA products containing a 3' poly(U) or poly(G) trailer sequence can completely inhibit the enzyme unlike poly(C). These results demonstrate that the eukaryotic and prokaryotic enzymes use different mechanisms for binding and recognizing pre-tRNA substrates.PhDBiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124656/2/3150187.pd