Gene expression depends on the function of numerous RNA processing enzymes, and their dysfunction or mis-regulation is often associated with disease. A hallmark of RNA processing endonucleases (such as RNase E, P, III, Cas9 and a host of others) is the ability to act on a large number of different RNA substrates in the cell despite variation from optimal sequence motifs in their binding sites. A key example is ribonuclease P (RNase P), a ubiquitous and essential RNA processing enzyme with a primary role in 5' end maturation of tRNAs. However, there is ample evidence that bacterial RNase P contributes to regulation of tRNAs, mRNAs, and other small RNAs; yet, we lack a basic understanding of how it is integrated into RNA metabolism. Even less is known regarding the specificity and RNA targets of the more structurally complex human RNase P enzyme. In the next five years we aim to define the roles of E. coli RNase P in RNA biosynthesis and regulation by comprehensively identifying its RNA substrates and cleavage sites using transcriptome-wide analysis tools. We will use new high throughput biochemical methods we developed in our lab to learn how variation from optimal sequence motifs affects RNase P processing rates. We will extend these studies to investigate human RNase P specificity and align the data analysis with our studies of bacterial RNase P. Comparison of these results with the emerging model derived from analysis of in vivo RNase P target sites will reveal the extent to which the intrinsic biophysical properties of RNase P are predictive of its functional specificity in vivo. Discontinuities between the in vitro and in vivo specificity models will be targeted for deeper investigation since they are likely to represent interesting departure points for discovering novel RNA biology. In parallel, we are determining how the active sites of RNases stabilize reaction transition states in order to accomplish catalysis. It is well-established that in solution RNA phosphoryl transfer reactions can occur either by step-wise or concerted mechanisms that further vary with respect to protonation, bonding, and charge distribution of the transition state. The intrinsic plasticity of phosphoryl transfer mechanisms raises questions central to enzymology: how do the active sites of enzymes alter reaction transition states?; and, do RNases and ribozymes, that catalyze the same chemical reaction, but with profoundly different active sites, stabilize the same transition states? We are addressing these questions by employing kinetic isotope effect (KIE) analyses to evaluate proposed mechanistic scenarios for RNases and ribozymes. The information gained will have broad impact by helping improve computational methods, facilitating the design of novel catalysts, and revealing the potential for development of transition state based inhibitors.
Our research aims to understand at a chemical level the two defining features of biological catalysis: exquisite substrate specificity and enormous rate enhancement. Our experiments center on enzymes essential for biosynthesis and regulation of RNA. The results provide basic information for designing novel catalysts and enzyme inhibitors as potential therapeutics to treat human diseases.