Poster abstracts

Poster number 107 submitted by Catherine Wilhelm

Structural and kinetic studies of a minimal protein-only RNase P

Catherine A. Wilhelm (Department of Chemistry at the University of Michigan), Kipchumba J. Kaitany (Department of Chemistry at the University of Michigan), Leena Mallik (Center for Structural Biology at the University of Michigan), Markos Koutmos (Department of Chemistry at the University of Michigan)

Abstract:
RNase P enzymes are responsible for pre-tRNA processing and maturation [1] across all domains of life. While the vast majority of RNase P enzymes require an RNA co-factor, Protein-Only RNase P enzymes (PRORPs) are found in human mitochondria and all tRNA-encoding compartments of plants[2]. PRORPs canonically consist of a pentatricopeptide repeat (PPR) RNA recognition domain, as well as a catalytic metallonuclease domain[3]. PRORPs were long thought to be the minimal RNase P construct, until the discovery of a further truncation in Aquifex aeolicus consisting of only the metallonuclease domain[4]. As these Homologs of Aquifex aeolicus RNase P (HARPs) lack the canonical PPR substrate recognition domain, we aim to characterize the substrate recognition and specificity of HARP enzymes utilizing a combination of electrophoretic mobility shift binding assays (EMSAs), single turnover assays, x-ray crystallography, and cryo-EM. Interestingly, our current structural data suggests that HARPs form a hexamer ring of six homodimers with twelve hypothetical active sites. Thus, we seek to determine the binding stoichiometry of HARPs, with preliminary data indicating that HARPs undergo multiple binding events and can specifically cleave multiple species of pre-tRNA without off-target activity. Furthermore, we seek to determine the observed rate constant of pre-tRNA cleavage and observe how active site mutants contribute to catalysis and substrate binding.

References:
[1] Altman and Smith. Nature 1971; 233:35-39.
[2] Daniels et al. RNA 2019; 25:286-291.
[3] Gobert et al. Nat Comm 2013; 4:1353.
[4] Nickel et al. PNAS 2017; 114(42):11121-11126.

Keywords: tRNA processing, RNA-binding proteins, protein structure