Poster abstracts

Poster number 50 submitted by Daina Zeng

Kinetic Characterization of Mature tRNA Release from B. subtilis RNase P Holoenzyme by using 3’ and 5’ end Fluorescein labeled pre-tRNA by Fluorescence Stopped-Flow Techniques

Daina Zeng (Department of Chemistry, University of Michigan, Ann Arbor, Michigan.), Nicole Hewlett (Department of Chemistry, University of Michigan), John Hsieh (Department of Chemistry, University of Michigan), Carol A. Fierke (Department of Chemistry, University of Michigan)

Abstract:
RNase P is an enzyme that catalyzes the 5' maturation of precursor tRNA, generating a mature tRNA and a short 5' leader RNA. Specifically, RNase P derived from Bactillus subtilis contains P RNA which is the catalytically active RNA component and a small protein cofactor known as P protein. Our lab has been researching the kinetic mechanism for this enzyme by fluorescence FRET stop flow techniques. The product release is rate limiting in steady-state conditions for the cleavage reaction catalyzed by both PRNA and the RNase P holoenzyme. In PRNA catalyzed reaction, the leader is released before the mature tRNA. However, in the holoenzyme-substrate complex, the leader may interact with the P protein and the mature tRNA with the P RNA which may result in a different product release sequence.

To study this question, we have directly monitored the dissociation rate constants for the two products from the RNase P holoenzyme: 5' leader and the mature tRNA during single turnover reaction to see which end disassociates faster to differentiate between at least two different possible pathways for product dissociation. We have prepared pre-tRNA substrates with fluorescence labeling at either 5' leader or the 3' tRNA moiety and measured the dissociation rate constants of both products by fluorescence stop flow techniques. Side-by-side comparison between the product release rates for the 5' leader and the mature tRNA for the RNase P holoenzyme will be studied also as a function of the leader length to understand the effects of protein-leader interaction on product dissociation kinetics.

Keywords: RNase P, Transient kinetics, Fluorescence