Poster abstracts
Poster number 152 submitted by Hong Duc Phan
Use of chemical modification and tandem mass spectrometry to identify RNA-protein contact sites in archaeal RNase P
Hong Duc Phan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210), Andrew Norris (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Tien-Hao Chen (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210)
Abstract:
RNase P is a ribonucleoprotein (RNP) that catalyzes cleavage of the 5ʹ-leader in precursor tRNAs. While RNase P is present in all domains of life, its composition varies between Bacteria, Archaea, and Eukarya. In Archaea, RNase P is composed of a catalytic RNA subunit (RNase P RNA; RPR) and up to five proteins (RNase P proteins; RPPs), including RPP21, RPP29, RPP30, POP5, and L7Ae. In vitro reconstitution studies have demonstrated that the RPP21-RPP29 binary complex enhances substrate affinity and the POP5-RPP30 binary complex increases the RPR’s cleavage rate. L7Ae has been found in other RNPs, and its functional role in archaeal RNase P remains to be defined. Although structures of the RPPs are available, the RNA-contacting residues and the dynamic changes that accompany assembly of the RPPs to form the RNase P complex have not been characterized. Little is known about the binding modes of the RPPs, except for L7Ae that binds an RNA structural motif called a kink-turn. In this study, we sought to covalently modify, either in the absence or presence of the RPR, the abundant lysine residues in RPPs and detect the modification sites by bottom-up tandem mass spectrometry (MS). To separate the RNase P RNP sub-assemblies from free RPPs, we engineered a sequence at the 5’-end of the RPR and used a biotinylated DNA oligonucleotide that is complementary to this sequence to pull-down the RNase P assembly using streptavidin-coated magnetic beads. The advantage of this MS-based approach over other higher resolution structural techniques is that its high-throughput allows examination of all the RNase P sub-assemblies en route to the final holoenzyme. Using archaeal RNase P as a model, we demonstrate the utility of chemical modification, affinity purification, and tandem MS to identify RNA-contacting residues on proteins in multi-subunit RNP complexes.
Keywords: RNase P, mass spectrometry, chemical modification