Ribonuclease P Catalysis:Metal Ion Interaction and Conformational Dynamics

[摘要] RNase P is responsible for catalyzing the 5’-end maturation of precursor tRNAs (pre-tRNAs). The catalytic RNA subunit of bacterial RNase P is essential for activity. Bound divalent metal ions and conformational dynamics in RNase P are important for enhancing the substrate affinity and contributing to the catalytic function. In this dissertation research, first a putative metal ion binding site in Bacillus subtilis RNase P RNA (PRNA) was examined using atom substitutions. The data suggest that the carbonyl oxygen at O6 position of G379 likely forms an outer-sphere contact with a metal-bound water that is important for substrate affinity and efficient catalysis. Secondly, R1ρ relaxation dispersion NMR experiments were used to probe the existence of alternate conformers in a P4 helix stem-loop mimic. The data revealed a shared transient state (TS) in the helix and an independent TS for U7.A computational MCSYM-LARMORD approach predicted a 3D model of the TS. In the shared TS, the A-tract region unwinds and G6 changes from anti conformation to syn that is coupled to the formation of a kink in the backbone between A5 and G6 that creates an electrostatic patch favorable for metal ion association. Additionally, the bulged U7 flips in and out of the helix with a motion that is faster than the dynamics of the shared TS.Mutants and nucleoside modifications were incorporated into the P4 helix in the full-length PRNA to examine the conformational change in the RNase P catalytic cycle. The kinetic data show strong correlations with the NMR results demonstrating that the TS observed in the P4 stem-loop mimics the P4 helix conformational change in RNase P. The likely functional role of the structural rearrangement in the P4 helix is the formation of an electrostatic site at the backbone between A49 and G50, which is favorable for Mg2+ ion binding to RNase P and activating catalysis. Overall, this work provides structural information about the metal ion binding sites and conformational change in the P4 helix during RNase P catalysis, and enhances our understanding of the functional roles of metal ion binding and conformational dynamics in RNA.

[发布机构] University of Michigan

[效力级别] Metal Ion Interaction

[关键词] RNase P;Metal Ion Interaction;Conformational Change;RNA dynamics;Biological Chemistry;Science;Chemistry