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

Author:

Marathe Ila A123ORCID,Lai Stella M234ORCID,Zahurancik Walter J23ORCID,Poirier Michael G235ORCID,Wysocki Vicki H234ORCID,Gopalan Venkat123ORCID

Affiliation:

1. Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA

2. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA

3. Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA

4. Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University, Columbus, OH 43210, USA

5. Department of Physics, The Ohio State University, Columbus, OH 43210, USA

Abstract

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-RPRC domain and Cy5-RPRS 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.

Funder

National Institutes of Health

Ohio State University Comprehensive Cancer Center

NIH

James Comprehensive Cancer Center

Behrman Research Fund

Publisher

Oxford University Press (OUP)

Subject

Genetics

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