The P4 Helix of RNase P: Structure, Dynamics, and Metal Binding by NMR.

Abstract

RNase P (ribonuclease P) is an enzyme that catalyzes hydrolysis of phosphodiester bonds in precursors of transfer RNA (tRNA) to form mature tRNA and that is found in all forms of life. In bacteria, the enzyme is composed of a large RNA (350-400 nt) component and a smaller (14 kDa) protein component. It requires Mg2+ for folding and catalysis, but the locations of the bound metals and their roles in structure and catalysis have not yet been deciphered. Numerous studies suggest that the highly conserved P4 helix located at the junction of the catalytic and specificity domains plays essential structural, dynamical and metal binding roles in catalysis. In this study we first characterized the structure, dynamics and metal binding properties of P4 through a combination of NMR techniques on an isolated stem-loop; we then established how these properties correlate with RNase P catalysis via mutagenesis measuring the effects of these structural changes on the in vitro activity of the holoenzyme. Our results reveal that the P4 sequence alone codes for a combination of local and global motions. The two helices undergo small amplitude domain motions around a flexible pivot point centered in and above the highly conserved uridine bulge which asymmetrically destabilizes the C-G Watson-Crick base-pair above it. Mg2+ ions bind to the distorted major groove in P4 in a region near the locally flexible pivot point for helix motions. Mg2+ binding does not significantly affect the structural dynamics of P4. A combination of comparative chemical shift titrations with different metals and Mn2+ paramagnetic relaxation enhancement provide evidence that Mg2+ ions associate with tandem guanines above the uridine bulge likely via inner-sphere interactions in the stem-loop construct. Swapping one of the C-G base-pairs with a G-C counterpart significantly alters the Mg2+ binding mode and dynamical properties of P4 without altering its overall structure. In in vitro single turnover assays catalyzed by the holoenzyme RNase P, this mutation decreases the catalytic rate constant by 60-fold compared to wild-type. These results expose sequence specific dynamical and metal binding properties in P4 that are likely important for RNase P assembly and catalysis.