Poster abstracts
Poster number 72 submitted by Stella Lai
A Tale of Two Types of Archaeal RNase P: Of Rate-Limiting Steps and Subunit Stoichiometries
Stella M. Lai (Department of Chemistry and Biochemistry, Center for RNA Biology, and Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University), Walter J. Zahurancik (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Center for RNA Biology, and Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University)
Abstract:
RNase P is an essential housekeeping enzyme that catalyzes the Mg2+-dependent 5′-maturation of precursor (pre)-tRNAs in all three domains of life. Defective tRNA processing in animals has been associated with a variety of diseases, including neurodegeneration, which underscores the need for a mechanistic understanding of RNase P assembly and catalysis. The canonical form of RNase P is a ribonucleoprotein (RNP) complex that is exemplified by archaeal RNase P, which consists of one catalytic RNA (RNase P RNA; RPR) and up to five protein subunits (RNase P proteins; RPPs): POP5, RPP21, RPP29, RPP30, and L7Ae. POP5•RPP30 and RPP21•RPP29 function as binary complexes to enhance the cleavage rate and improve substrate binding, respectively. However, the role of monomeric L7Ae, which binds RNA structural modules called kink-turns (K-turns) to induce axial bending, is unclear. We recently reported that L7Ae binds three K-turns in type A [e.g., Pyrococcus furiosus (Pfu)] and two K-turns in type M [e.g., Methanocaldococcus jannaschii (Mja), Methanococcus maripaludis (Mma)] euryarchaeal RNase P, which are classified based on the secondary structure of their RPRs. To determine whether the number and location of the K-turns affect the ability of L7Ae to contribute directly to RNase P catalysis, we are using rapid quench-flow-based single-turnover and manual quench-based multiple-turnover assays to measure the kinetics of pre-tRNA cleavage by Pfu, Mja, and Mma RNase P assembled in the absence and presence of L7Ae. In parallel, we are using native mass spectrometry to study the composition and stoichiometry of these RNase P assemblies to establish a structural context for the biochemical results. Together, these complementary analyses provide valuable insights into protein-aided catalysis in this ancient, multi-subunit RNP.
Keywords: RNase P, native mass spectrometry, tRNA processing