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

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. B. subtilis ribonuclease P (RNase P), composed of a catalytically active RNA (PRNA) and a small protein (P protein) subunit, 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 (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 FRET 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 Å closer to the PRNA-P protein interface during the ES to ES* transition and suggest that the metal-dependent conformational change re-organizes the bound substrate in the active site to form a catalytically competent ES* complex.