Poster abstracts

Poster number 49 submitted by John Hsieh

Conformational Change in the B. subtilis RNase P Holoenzyme•pre-tRNA Complex is Induced by Binding of a High-affinity Inner-sphere Divalent Ion

John Hsieh (Department of Chemistry, University of Michigan, Ann Arbor, Michigan.), David Rueda (Department of Chemistry, Wayne State University, Detroit, Michigan.), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, Michigan.), Carol A. Fierke (Department of Chemistry, University of Michigan, Ann Arbor, Michigan.)

Abstract:
Ribonuclease P holoenzyme (RNase P) catalyzes the 5' maturation of precursor tRNAs (pre-tRNAs). The B. subtilis RNase P holoenzyme consists of an RNA component and a small protein component. The RNA component of RNase P alone is catalytically active in vitro; however the protein cofactor is essential for activity under physiological conditions. The protein component directly interacts with the 5' leader of the pre-tRNA substrate and enhances metal affinity. While metal ions capable of inner-sphere coordination are not essential for formation of the RNase P holoenzyme•pre-tRNA complex, inner-sphere coordination of metal ions is essential for catalytic activity. Defining the roles of metal ions in catalysis by RNase P holoenzyme is essential to understand its mechanism.

We are using fluorescence titration and transient kinetics to probe the thermodynamics and kinetics of binding Ca(II) to the RNase P holoenzyme•pre-tRNA (ES) complex, and time-resolved FRET to characterize structural alterations in a Ca(II)-induced conformational change in this complex. In 2 mM Co(NH₃)₆³, 189 mM KCl, and 50 mM Tris/MES, pH 6.0, addition of 100 μM CaCl₂ enhances the binding affinity of pre-tRNAAsp for B. subtilis RNase P holoenzyme by 3-4 fold. Fluorescence stopped-flow and trFRET data suggest that Ca(II) binding induces a conformational change in the ES complex. Addition of similar concentrations of Mg(II) also induces this conformational change, but does not significantly enhance cleavage activity. These data suggest that formation of the RNase P holoenzyme•pre-tRNA complex creates a high affinity, inner sphere binding site for Ca(II) or Mg(II). Furthermore, metal binding is coupled to a conformational change that enhances substrate binding affinity and organizes the active site in the ES complex. However, one or more additional Mg(II) ions are required to activate catalytic activity.

Keywords: RNase P, conformational change, metals