Poster abstracts

Poster number 22 submitted by John Hsieh

Conformational changes in B. subtilis RNase P holoenzyme associated with substrate binding measured by transient fluorescence techniques

John Hsieh (Department of Chemistry, University of Michigan), Daina Zeng (Department of Chemistry, University of Michigan), Carol A. Fierke (Departments of Chemistry and Biological Chemistry, University of Michigan)

Abstract:
Ribonuclease P (RNase P) catalyzes the 5’ maturation of precursor tRNA, generating a mature tRNA and a short 5’ leader RNA. The Bactillus subtilis RNase P contains a catalytically active RNA component and a small protein cofactor known as P protein, and both components contribute to molecular recognition. To further evaluate the catalytic mechanism, we have developed a complete kinetic scheme for the B. subtilis RNase P using transient kinetic techniques. We measured the association and dissociation rate constants by mixing RNase P with pre-tRNA substrates containing a fluorescent label at either the 5’ leader or the 3’ tRNA moiety and measured the time-dependent changes in fluorescence by stopped-flow techniques. We also measured the chemical cleavage rate constant using chemical quench flow techniques. Under single-turnover conditions ([E] >> [S]), we have observed two phases in the binding kinetics: a rapid, diffusion-controlled association step followed by a step with hyperbolic dependence on holoenzyme concentration where the limiting rate constant is faster than the cleavage rate constant. This indicates that a conformational change in the holoenzyme-substrate complex precedes the cleavage step. This step is also facilitated by the addition of divalent cations. In addition, the dissociation rate constants from the E•tRNA•leader complex are identical for both tRNA and the 5’ leader sequence suggesting that a rate-limiting conformational change step also precedes release of both products. This is different than the kinetic scheme for the PRNA-catalyzed reaction where the 5’ leader dissociates before the mature tRNA. This difference is likely due to the interaction of the pre-tRNA leader with the P protein in the holoenzyme.

Keywords: RNase P, transient kinetics, enzyme mechanism