Poster abstracts

Poster number 165 submitted by Walter Zahurancik

Use of single-molecule fluorescence co-localization spectroscopy to study archaeal RNase P assembly

Walter J. Zahurancik (Chemistry and Biochemistry, The Ohio State University), Khan L. Cox (Physics, The Ohio State University), Ila A. Marathe (Microbiology, The Ohio State University), Stella M. Lai, Lien B. Lai, Xiao Ma, Vaishnavi Sidharthan, Mark P. Foster (Chemistry and Biochemistry, The Ohio State University), Michael G. Poirier (Physics, The Ohio State University), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University)

Abstract:
RNase P is an essential enzyme that catalyzes the 5′ maturation of precursor (pre)-tRNAs in all three domains of life. The ribonucleoprotein (RNP) form of RNase P is comprised of a single catalytic RNase P RNA (RPR) and a variable number of RNase P proteins (RPPs): one in Bacteria, up to five in Archaea, and up to ten in Eukarya. Given its biochemical tractability in vitro and its homology to the eukaryotic enzyme, archaeal RNase P is a useful model for studying the role of proteins in multi-subunit RNPs. While a recent cryo-electron microscopy structure has provided critical insights into the subunit organization of a functional archaeal RNase P holoenzyme1, the enzyme’s assembly pathway and its subunit composition en route to the final, higher-order RNP remain unclear. We recently reported that there are three kink-turns in the Pyrococcus furiosus (Pfu) RPR and used native mass spectrometry to show that up to three copies of L7Ae bind to the RNA2. Here, we use single-molecule fluorescence (SMF) co-localization spectroscopy to validate the number of copies of L7Ae that bind to the Pfu RPR by monitoring the binding of Cy5-labeled Pfu L7Ae to immobilized, Cy3-labeled Pfu RPR. Step-wise changes in Cy5 fluorescence reflect the sequential association and dissociation of multiple L7Ae copies to a single RPR molecule. Our goal is to use this SMF co-localization spectroscopy approach to study the binding kinetics of the other RPPs and to gain insights into the hierarchy of archaeal RNase P assembly.

References:
1. Wan F, Wang Q, Tan J, Tan M, Chen J, Shi S, Lan P, Wu J, Lei M. (2019) Cryo-electron microscopy structure of an archaeal ribonuclease P holoenzyme. Nat. Commun., 10: 2617.
2. Lai LB, Tanimoto A*, Lai SM*, Chen W-Y, Marathe IA, Westhof E, Wysocki VH, Gopalan V. (2017) A novel double kink-turn module in euryarchaeal RNase P RNAs. Nucleic Acids Res., 45: 7432-7440. *joint second authors

Keywords: RNase P, Ribozyme, Single-molecule fluorescence