Poster number 115 submitted by Catherine Wilhelm
Structural and kinetic characterization of multiple oligomeric states in a minimal protein-only RNase P
Catherine A. Wilhelm (Department of Chemistry, University of Michigan), Abigail L. Kelly, Shayna B. Brotzman, Johnny Mendoza, Suada Leskaj (Department of Chemistry, University of Michigan), Leena Mallik (Center for Structural Biology, Life Sciences Institute, University of Michigan), Markos Koutmos (Departments of Chemistry and Biophysics, University of Michigan)
Transfer RNA (tRNA) genes are often transcribed as precursor tRNAs (pre-tRNAs) with additional leader and trailer sequences. In one of the first steps of the extensive processing to convert pre-tRNA to mature tRNA, RNase P enzymes cleave the 5’-end of pre-tRNA in all domains of life. Homologs of Aquifex RNase P (HARPs) are a newly discovered class of RNase P consisting of only a metallonuclease domain. HARPs are minimal proteins compared to their ribonucleoprotein (RNP) and protein-only RNase P (PRORP) counterparts and they appear to lack the usual substrate recognition domain. Despite these features, HARPs maintain the ability to bind to and cleave the 5’ leader sequence of tRNAs.
To improve our understanding of how this minimal RNase P enzyme, we used a combination of x-ray crystallography, cryo-EM, and biochemical assays. In this work, we solved the cryo-EM structure of A. thaliana HARP, which is a ring-like hexamer of dimers, and the crystal structure of H. thermophilus HARP, a ring-like heptamer of dimers. These structures reveal the active site is approximately 42 Å away from a helical region of positively charged residues – the perfect distance for the acceptor stem of a pre-tRNA. Curious about the potential for multiple oligomeric states of HARP, we used native ion-mobility mass spectrometry to identify multiple oligomeric states in solution we had previously not detected. We also discovered that HARPs bind pre-tRNAs in multiple oligomeric states, likely as both a tetramer and a dodecamer. To round out our studies, we determined that HARPs specifically cleave and bind pre-tRNA from multiple species with activity similar to other PRORPs. This data provides a closer look at the similarities in tRNA binding recognition and processing across disparate classes of RNase P.
Nickel et al. (2017) Minimal and RNA-free RNase P in Aquifex aeolicus PNAS 114(42) 11121-11126.
Feyh et al. (2021) Structure and mechanistic features of the prokaryotic minimal RNase P eLife 10:e70160.
Keywords: RNase P, tRNA, RNA processing