# Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes

> **NIH NIH R35** · UNIVERSITY OF FLORIDA · 2022 · $323,384

## Abstract

PROJECT SUMMARY/ABSTRACT
 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.

## Key facts

- **NIH application ID:** 10434828
- **Project number:** 5R35GM127100-05
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** MICHAEL E. HARRIS
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $323,384
- **Award type:** 5
- **Project period:** 2018-07-05 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10434828

## Citation

> US National Institutes of Health, RePORTER application 10434828, Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes (5R35GM127100-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10434828. Licensed CC0.

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