Poster abstracts
Poster number 36 submitted by Duc Phan
Use of native mass spectrometry and mass photometry to elucidate structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein
Hong Duc Phan (Department of Chemistry and Biochemistry,The Ohio State Biochemistry Program,Center for RNA Biology), Andrew S. Norris (Department of Chemistry and Biochemistry,Resource for Native Mass Spectrometry-Guided Structural Biology), Chen Du (Department of Chemistry and Biochemistry,Resource for Native Mass Spectrometry-Guided Structural Biology), Kye Stachowski, Mark P. Foster (Department of Chemistry and Biochemistry,Center for RNA Biology), Vicki H. Wysocki (Department of Chemistry and Biochemistry,The Ohio State Biochemistry Program,Resource for Native Mass Spectrometry-Guided Structural Biology), Venkat Gopalan (Department of Chemistry and Biochemistry,The Ohio State Biochemistry Program,Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology)
Abstract:
RNase P is a ribonucleoprotein (RNP) complex that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM structure of the Methanocaldococcus jannaschii (Mja) RNase P holoenzyme revealed unexpectedly a dimeric configuration, with each monomer of this archaeal variant containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, and L7Ae). Here, we used native mass spectrometry, mass photometry, and biochemical activity assays to (i) validate the oligomeric state of the Mja RNase P holoenzyme, (ii) report on a different stoichiometry for each holoenzyme monomer, where up to two copies of L7Ae are present, and (iii) identify the copies of L7Ae necessary for optimal cleavage activity. Moreover, by mutating all kink-turns (the binding sites for L7Ae) in the RPR, we made the surprising discovery that abolishing L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the safeguard provided by protein-protein interactions between L7Ae and other RPPs. Collectively, our results provide new insights into the architecture and evolution of RNase P, and highlight the value of using native MS and MP to augment the information obtained from low/medium-resolution cryo-EM structures.
Keywords: RNase P, RNA, mass spectrometry