title: A divalent cation stabilizes the active conformation of the B. subtilis RNase P x pre-tRNA complex: a role for an inner-sphere metal ion in RNase P.
abstract: Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5'-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P x pre-tRNA (enzyme-substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES* conformer) before the cleavage step. Here, we show that the ES* conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor-acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5' leader. These data demonstrate that the 5' leader of pre-tRNA moves 4 to 6 A closer to the PRNA x P protein interface during the ES-to-ES* transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES* complex.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: John
authors.initials: J
authors.lastname: Hsieh
authors.forename: Kristin S
authors.initials: KS
authors.lastname: Koutmou
authors.forename: David
authors.initials: D
authors.lastname: Rueda
authors.forename: Markos
authors.initials: M
authors.lastname: Koutmos
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry*; D004795:Enzyme Stability; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002413:Cations, Divalent; D008670:Metals; D012322:RNA Precursors; D043262:Ribonuclease P