Divergent molecular assembly and catalytic mechanisms between bacterial and archaeal RNase P in pre-tRNA cleavage.

Abstract

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from <i>E. coli</i> (<i>Escherichia coli</i>) and <i>Mja</i> (<i>Methanocaldococcus jannaschii</i>) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between <i>E. coli</i> and <i>Mja</i> at both the RNA and protein levels. Specifically, the RNA of <i>E. coli</i> RNase P (<i>Eco</i>RPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of <i>E. coli</i> RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, <i>Mja</i> RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of <i>Mja</i> RPR. Mutation experiments suggest that the functional deficiency of <i>Mja</i> RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for <i>Mja</i> RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms.