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.
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.
Hsieh
John
J
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Koutmou
Kristin S
KS
Rueda
David
D
Koutmos
Markos
M
Walter
Nils G
NG
Fierke
Carol A
CA
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
D002413:Cations, Divalent; D008670:Metals; D012322:RNA Precursors; D043262:Ribonuclease P