A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P . Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P

JOURNAL OF MOLECULAR BIOLOGY

卷:400

Article

Hsieh, John¹  Koutmou, Kristin S.¹  Rueda, David¹  Koutmos, Markos²  Walter, Nils G.¹  Fierke, Carol A.^1,3 

[1] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA

[2] Univ Michigan, Inst Life Sci, Ann Arbor, MI 48109 USA

[3] Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USA

关键词: RNase P;    tRNA processing;    conformational change;    metals;    trFRET;   

DOI  :  10.1016/j.jmb.2010.04.050

来源: Elsevier

【 摘 要 】

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.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 Forster 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 angstrom closer to the PRNA.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. (C) 2010 Elsevier Ltd. All rights reserved.

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