Poster abstracts

Poster number 25 submitted by Zoe Limberopoulos

Characterizing the 3' Cleavage Activity of Aquifex aeolicus RNase P Homologs on pre-tRNA Substrates

Zoe Limberopoulos (University of Michigan, Chemistry), Daniel J. Corey (University of Michigan, Chemistry), Matthew Yacoub (University of Michigan, Chemistry), Catherine A. Wilhelm (University of Michigan, Cellular and Developmental Biology), Markos Koutmos (University of Michigan, Chemistry and Biophysics)

Abstract:
RNase P is a conserved enzyme across all three domains of life, responsible for cleavage of 5’ leader nucleotides from precursor tRNAs (pre-tRNAs) with the assistance of a magnesium cation cofactor. This cleavage is a critical step in tRNA maturation. Homologs of Aquifex aeolicus RNase P enzymes (HARPs) are the newest class of RNase P enzymes discovered, which lack the pentatricopeptide repeat RNA recognition domain found in most other Protein-Only RNase P enzymes (PROPs). Our lab previously studied the HARP enzyme from H. thermophilus, during which data collected by us and others suggested that HARPs may cleave multiple species of pre-tRNA, as well as undergo multiple binding events. Additionally, we have expanded into another organism, Aquifex aeolicus, to study pre-tRNA cleavage of the 3’ trailer ends of pre-tRNA by both Ht and Aa HARPs, providing evidence for a novel method of pre-tRNA maturation in bacteria. These results expand our understanding of HARP substrate recognition and catalytic scope, while providing insight into how several factors including temperature, magnesium concentration, and substrate length may influence HARP cleavage activity.

References:
(1) Wu Meyers, N.; Karasik, A.; Kaitany, K.; Fierke, C. A.; Koutmos, M. Gambogic Acid and Juglone Inhibit RNase P through Distinct Mechanisms. J. Biol. Chem. 2022, 298 (12), 102683. https://doi.org/10.1016/j.jbc.2022.102683.​

(2) Wilhelm, C. A.; Mallik, L.; Kelly, A. L.; Brotzman, S.; Mendoza, J.; Anders, A. G.; Leskaj, S.; Castillo, C.; Ruotolo, B. T.; Cianfrocco, M. A.; Koutmos, M. Bacterial RNA-Free RNase P: Structural and Functional Characterization of Multiple Oligomeric Forms of a Minimal Protein-Only Ribonuclease P. J. Biol. Chem. 2023, 299 (11), 105327. https://doi.org/10.1016/j.jbc.2023.105327.​

(3) Smith, D.; Pace, N. R. Multiple Magnesium Ions in the Ribonuclease P Reaction Mechanism. Biochemistry 1993, 32 (20), 5273–5281. https://doi.org/10.1021/bi00071a001.

Keywords: RNA, pre-tRNA maturation, HARP