Poster abstracts

Poster number 86 submitted by Xin Ma

Determination of the stoichiometry of Pyrococcus furiosus RNase P by native mass spectrometry

Xin Ma (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Lien B. Lai (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Vicki Wysocki (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University)

Abstract:
RNase P is an essential ribonucleoprotein (RNP) enzyme that cleaves the 5’-leader of precursor tRNAs in a Mg²⁺-dependent manner to produce functional mature tRNAs in all three domains of life. RNase P present in the thermophilic archaeon Pyrococcus furiosus contains one catalytic RNA (abbreviated RPR for RNase P RNA) and five protein cofactors (RPPs) called POP5, RPP21, RPP29, RPP30, and L7Ae. RNase P activity (of varying potency) can be demonstrated in the holoenzyme reconstituted in vitro using the RPR with either or both interacting RPP pairs (POP5−RPP30 or/and RPP21−RPP29) and with all five RPPs. Previous studies have shown that (1) these RPP pairs can be co-expressed and co-purified from Escherichia coli, (2) POP5−RPP30 forms a tetramer, and (3)RPP21−RPP29 forms a dimer (in the absence of the cognate RPR). However, the stoichiometry of the individual subunits in the reconstituted RNase P complex remains unknown. Native mass spectrometry, ion mobility and tandem mass spectrometry were employed to explore the interaction of the two RPP pairs by themselves, with each other and with the RPR. Our results confirmed that the two RPP pairs behave as reported previously, and additionally revealed a higher-order structure (POP52−RPP302.RPP21−RPP29). However, in the presence of the RPR, only one copy of each RPP is found in the holoenzyme complex. Moreover, we observed that surface induced dissociation (SID) can break up the holoenzyme (lacking L7Ae), while collision induced dissociation (CID) cannot do so effectively. Our studies collectively demonstrate the advantage of SID in successfully determining the composition of an RNP complex and provide insight into the assembly of archaeal RNase P.

Keywords: RNase P, mass spectrometry, stoichiometry