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

作者：Hsieh John; Koutmou Kristin S; Rueda David; Koutmos Markos; Walter Nils G; Fierke Carol A^*

来源：Journal of Molecular Biology, 2010, 400(1): 38-51.

DOI：10.1016/j.jmb.2010.04.050

• RNase P
• tRNA processing
• conformational change
• metals
• trFRET

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.

• 出版日期2010-7-2