Talk Abstracts

Talk on Friday 03:15-03:30pm submitted by Ila Marathe

Protein cofactors and substrate dictate Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Ila A. Marathe (Department of Chemistry and Biochemistry, Ohio State University), Walter J. Zahurancik (Department of Chemistry and Biochemistry, Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry, Ohio State University), Khan L. Cox, Michael G. Poirier (Department of Physics, Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, Ohio State University)

Abstract:
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ~250-500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ~10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Keywords: RNase P, Conformational dynamics, FRET