Poster abstracts

Poster number 122 submitted by Vibhuti Wadhwa

Use of pulsed EPR to probe protein cofactor-mediated long-range remodeling of the catalytic RNA in archaeal RNase P

Vibhuti Wadhwa (Department of Chemistry and Biochemistry, The Ohio State University), Xiao Ma (Department of Chemistry and Biochemistry, The Ohio State University), Antonia Duran (Ohio State Biochemistry Program, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
In all domains of life, RNase P is an essential enzyme required for processing 5’-leader sequence of precursor tRNAs to yield a mature tRNA. This universally conserved ribonucleoprotein form of RNase P consists of a single RNA subunit (RPR) and variable number of protein subunits (RPPs): one RPP in bacteria, up to five in archaea and up to ten in eukarya.1 Although the RPR is active on its own in vitro, the RPPs are essential in vivo. Biochemical reconstitution of the archaeal RPR with RPPs significantly improves the enzyme’s substrate affinity and cleavage rate.2,3 Archaeal RNase P serves as an excellent model to understand the need the for increased protein complexity in higher organisms, due to its tractability and homology of archaeal and eukaryotic RPPs.1 To probe the structural changes induced in the archaeal RPR upon binding of RPPs, we are using double electron-electron resonance (DEER), a pulsed EPR technique, that allows distance measurements between site-specifically spin labeled RPPs or RPR. Insights into the conformational rearrangement of the RPR as a function of RPPs will provide information about subunit positions and structural changes that are responsible for modulating enzymatic activity.

References:
(1) Lai, L. B., Vioque, A., Kirsebom, L. A., and Gopalan, V. (2010) Unexpected diversity of RNase P, an ancient tRNA processing enzyme: Challenges and prospects. FEBS Lett.
(2) Chen, W. Y., Pulukkunat, D. K., Cho, I. M., Tsai, H. Y., and Gopalan, V. (2010) Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex. Nucleic Acids Res. 38, 8316–8327.
(3) Cho, I.-M., Lai, L. B., Susanti, D., Mukhopadhyay, B., and Gopalan, V. (2010) Ribosomal protein L7Ae is a subunit of archaeal RNase P. Proc. Natl. Acad. Sci. U. S. A. 107, 14573–14578.

Keywords: RNase P, EPR, Spin label