The Multiple Substrate Recognition Properties of Ribonuclease P: Achieving Uniformity in Processing Kinetics Despite Variation in Substrate Structure

Abstract

Ribonuclease P is an essential enzyme that is responsible for catalyzing the maturation of the 5’ end of transfer RNA. In Bacteria, the RNase P holoenzyme is composed of a RNA subunit and a protein subunit. The RNA subunit of RNase P contains the enzyme active site and has the ability to process substrates in the absence of the protein component in vitro, but works as a holoenzyme in vivo. While nucleotide recognition elements adjacent to the site of processing have been identified, different tRNA sequences vary considerably. Remarkably, rates of pre-tRNA processing are uniform despite this variation in substrate sequence and structure. The mechanistic basis for multiple substrate recognition by the holoenzyme is the focus of this study, with the ultimate goal being a better understanding of uniformity in ptRNA processing and discriminates between cognate and non-cognate RNAs. We are determining kinetic schemes for a number of pre-tRNAs using fluorescence assays and standard discontinuous assays. Multiple turnover kinetics have been obtained for ptRNA608MET, a consensus pre-tRNA, and ptRNA605f-MET a non-consensus sequence. Both ptRNAs display a similar Vmax, and Km values. Neither tRNA displayed any burst nor lag phases in pre-steady state kinetics implying that the rate limiting step for our processing model is catalysis. Our initial hypothesis is that uniformity in substrate kcat/Km values results from differential 5’ leader sequence interactions with the protein that compensate for deviations from tRNA consensus recognition sequences. To obtain more insight into sequence and structure influence, we have set up a series of multiple turnover experiments with a consensus and non-consensus ptRNA competing against one another.