Substrate specificity, catalysis, and inhibition of bacterial ribonuclease P

Ribonuclease P (RNase P) occurs ubiquitously in biology and catalyzes the 5’ end maturation of tRNAs and other RNA precursors.  Bacterial RNase P has long been known to cleave multiple mRNAs and other small RNAs, although a complete accounting of its roles in gene regulation is unavailable. Despite the fact that the structure and catalytic function of RNase P are well studied, much less is known about its target sites in the transcriptome and potential roles in gene regulation.  RNase P provides a valuable system to learn how a highly specific RNase accommodates variation in sequence and structure among its targets in the transcriptome; and, to investigate how its RNA sequence and structure specificity are tuned to achieve required processing rates and accomplish regulation. Our research addresses these questions and in so doing provides information necessary to bridge the gap in our understanding between the biophysical behavior of RNase P, biological functions, and realize its potential as an antimicrobial target.
  • Zhao J, Harris ME. (2019). Distributive enzyme binding controlled by local RNA context results in 3′ to 5′ directional processing of dicistronic tRNA precursors by Escherichia coli ribonuclease P. Nucleic Acids Res. 47(3):1451-1467.
  • Niland CN, Anderson D, Jankowsky E and Harris ME. (2017). The contribution of the C5 protein subunit of Escherichia coli ribonuclease P to specificity for precursor tRNA is modulated by proximal 5′ leader sequences. RNA. 23(10):1502-1511.
  • Jankowsky E and Harris ME. (2017). Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing. Methods. Apr 15:118-119.
  • Jain N, Lin H-C, Harris ME and Tolbert B. (2017). Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution. Proc Natl Acad Sci. 114(9):2206-2211.
  • Lin H-C, Zhao J, Niland CN, Tran B, Jankowsky E and Harris ME. (2016). Comprehensive analysis of the C5 protein specificity landscape reveals RNA structure and sequence preferences that direct ribonuclease P substrate specificity. Cell Chem Biol. 23(10):1271-1281.
  • Jankowsky E and Harris ME. (2015). Specificity and non-specificity in RNA-protein interactions. Nat Rev Molec Cell Biol. 16(9):533-44.

Mechanistic enzymology of phosphoryl transfer enzymes including ribozymes

A fundamental principle of biology is that enzymes achieve catalysis by stabilizing reaction transition states. Knowledge of enzyme transition state structure provides deep insight into catalytic mechanism as well as providing design principles for potent inhibitors that are used as drugs to treat cancer, heart disease and combat infection. We are using kinetic isotope effect analyses combined with computational simulations to investigate the mechanisms of key phosphoryl transfer enzymes.
  • Yoon S, Harris ME. (2021). Beyond the Plateau: pL dependence of proton inventories as a tool for studying ribozyme and ribonuclease catalysis. Biochemistry. 60(37):2810-2823.
  • Bevilacqua PC, Harris ME, Piccirilli JA, Gaines C, Ganguly A, Kostenbader K, Ekesan S, York DM. (2019). ACS Chem Biol. 14(6):1068-1076.
  • Lu J, Koo SC, Weissman BP, Harris ME, Li NS, Piccirilli JA. (2018). Evidence that nucelophile deprotonation exceeds bond formation in the HDV ribozyme transition state. Biochemistry. 57(25):3465-3472.
  • Harris ME, York DM, Piccirilli JA, Anderson VA. (2017). Kinetic isotope effect analysis of RNA 2′-O-transphosphorylation.  Methods Enzymol. 596:433-457.
  • Zhang S, Gu H, Chen H, Strong E, Ollie EW, Kellerman D, Liang D, Miyagi M, Anderson VE, Piccirilli JA, York DM and Harris ME. (2016). Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+. Chem Comm. 52(24):4462-5.
  • Lee T-S, Radak BK, Harris ME and York DM. (2016). A two metal ion conformational switch pathway for HDV ribozyme activation. ACS Catalysis. 126, 1843-1853.
  • Zhang S, Gu H, Chen H, Strong E, Ollie EW, Kellerman D, Liang D, Miyagi M, Anderson VE, Piccirilli JA, York DM and Harris ME. (2016). Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+. Chem Comm. 52(24):4462-5.

Allosteric regulation and inhibition of human ribonucleotide reductase

Ribonucleotide reductase generated deoxynucleotides for DNA synthesis and regulates nucleotide pools. RR is a widespread and conserved enzyme that is a key experimental system for exploration of protein allostery, and an important chemotherapeutic target. Development of small molecule allosteric regulators and inhibitors of human RR is an important goal. In collaboration with the laboratory of Dr. Chris Dealwis we developed a protocol for identifying inhibitors of RR that combines in silico docking, biochemical assays of binding and activity, and cell toxicity studies. We successfully identified multiple compounds that target the different nucleotide binding sites and oligomerization interface on RR. In parallel, we have pursued studies to understand the structure and molecular dynamics that underlie allosteric communication. We identified the key functional groups on nucleotide effectors that drive communication of chemical information essential for allosteric regulation of substrate specificity as well as uncovering long range communication between the two different allosteric binding sites on RR.
  • Misko TA, Liu YT, Harris ME, Oleinick NL, Pink J, Lee HY, Dealwis CG. (2019). Structure-guided design of anti-cancer ribonucleotide reductase inhibitors.  J Enzyme Inhib Med Chem. 34(1):438-450.
  • Huff S, Ahmad MF, Yang M, Agrawal P, Pink J, Harris ME, Dealwis C, Viswanathan R (2018). Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase. J Med Chem. 61(3):666-680.
  • Knappenberger AJ, Grandhi S, Sheth R, Ahmad MD, Viswanathan R, and Harris ME (2017). Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase. J Biol Chem. 292(40):16463-16476.
  • Ahmad MF, Alam I, Huff SE, Pink J, Flanagan SA, Shewach D, Misko TA, Oleinick NL, Harte WE, Viswanathan R, Harris ME, and Dealwis CG. (2017). Potent competitive inhibition of human ribonucleotide reductase by a non-nucleoside small molecule. Proc Natl Acad Sci 114(31):8241-8246.
  • Knappenberger AJ, Ahmad FM, Dealwis CG and Harris ME. (2016). Nucleotide effector analogs allosterically regulate human ribonucleotide reductase and identify chemical determinants of substrate specificity. Biochemistry. 55(41):5884–5896.
  • Ahmad M, Huff S, Pink J, Alam I, Zhang A, Perry K, Harris ME, Misko T, Porwal SK, Oleinick NL, Viswanathan R, Miyagi M, Dealwis CG. (2015). Identification of non-nucleoside human ribonucleotide reductase inhibitors. J Med Chem. 58(24):9498-509.