Poster abstracts
Poster number 83 submitted by Ila Marathe
Use of FRET and SHAPE-Seq to elucidate structure-function relationships in archaeal RNase P, a multi-subunit catalytic ribonucleoprotein
Ila A. Marathe (Department of Microbiology, Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Paul D. Carlson, Molly E. Evans (Department of Chemical and Biological Engineering, Northwestern University), Stella M. Lai, Vicki H. Wysocki (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology, The Ohio State University), Michael G. Poirier (Department of Physics, The Ohio State University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
RNase P catalyzes the Mg2+-dependent 5'-maturation of tRNAs in all life forms. The ribonucleoprotein (RNP) form of RNase P consists of a catalytic RNase P RNA (RPR) and a variable number of RNase P Protein (RPP) cofactors: one in bacteria, and up to five in archaea and ten in eukaryotes. We use archaeal RNase P as a model to understand how multiple proteins independently and collectively modulate the functional scope of ribozymes, especially under physiological conditions. This objective gains significance due to an increased appreciation for the roles of RNPs in tissue complexity and human disease. Because the archaeal RPR alone requires ~500 mM Mg2+ even to support weak activity and needs RPPs for robust activity at physiological [Mg2+], we postulated that structural remodeling of the RPR by RPPs likely mirrors some of the changes promoted by high [Mg2+]. To test this hypothesis, we leveraged the powerful suite of RNase P assays, SHAPE-Seq, and FRET studies to identify functional and structural changes in the Pyrococcus furiosus (Pfu) RPR. First, we measured Pfu RPR’s activity in the presence of 10 to 500 mM Mg2+ to determine the [Mg2+] required for function. Second, we used SHAPE-Seq to interrogate the RPR at varying [Mg2+] and with single-nucleotide resolution to obtain insights into RPR structure (e.g., new inter-domain tertiary contacts). Finally, using dual-fluor-labeled Pfu RPR (currently in preparation), we plan to perform ensemble FRET experiments to determine the [Mg2+] required to establish long-range contacts and to use RPR mutants to validate any inter-domain contacts. This multi-pronged strategy helps parse the dual roles of Mg2+ in RNase P, wherein diffusely-bound Mg2+ promotes RPR structure and site-specific Mg2+ enables RPR catalysis. Collectively, our results suggest a model to link RPR structural changes to functional outcomes and establish a baseline to evaluate how archaeal RPPs forge a functional holoenzyme through concerted RPR remodeling.
Keywords: RNase P, RNA-protein interactions, Ribonucleoprotein assembly