Poster abstracts

Poster number 45 submitted by Wen-Yi Chen

Use of archaeal RNase P as a model system to elucidate the functional interplay among subunits in a catalytic RNP complex

Wen-Yi Chen (Molecular Cellular Developmental Biology Program), Yiren Xu (Ohio State Biochemistry Program), I-Ming Cho (Molecular Genetics), Mark P. Foster (Department of Biochemistry), Venkat Gopalan (Department f Biochemistry)

Abstract:
Ribonuclease P (RNase P) is a Mg²⁺-dependent endoribonuclease which catalyzes the 5' maturation of precursor tRNAs (ptRNAs). Although its primary function is conserved in all three domains of life, the subunit make-up of this ribonucleoprotein (RNP) varies. In bacteria, the holoenzyme is made up of one RNase P RNA (RPR) and one RNase P protein (RPP). In eukarya, RNase P contains an RPR and at least nine RPPs. Intermediate in complexity, archaeal RNase P comprises an RPR and four RPPs, which are homologs to their to eukaryal counterparts. We employ archaeal RNase P as a model to address the roles of multiple RPPs in archaeal/eukaryal RNase P catalysis. By using recombinant Methanothermobacter thermautotrophicus (Mth) RPR and four RPPs (termed POP5, RPP21, RPP29, and RPP30), we have reconstituted the holoenzyme and performed single-turnover (STO) measurements. The maximal rate of ptRNA cleavage by the holoenzyme (i.e., RPR + 4RPPs) is ~60-fold faster than that for the catalytic RPR alone. Using functional binary RPP complexes, we established that POP5-RPP30 is solely responsible for enhancing the rate of the archaeal RPR, suggesting it directly aids the phosphodiester bond-breaking step. Conversely, RPP21-RPP29 lowers the K_M(STO) by 16-fold indicating its central role in increasing the RPR's affinity for the ptRNA substrate. Both binary RPP complexes lower the RPR’s requirement for monovalent and divalent ion concentrations. Lastly, our findings that (i) a mutant Mth RPR with weakened binding of active-site Mg²⁺ is rescued upon addition of RPPs, and (ii) an N-terminal deletion mutant of RPP29 that fails to bind RPP21 (as judged by ITC and NMR experiments) is functional upon addition of RPR, illustrate the cooperative subunit interactions critical for driving RNase P towards its functional conformation. Together, these studies are beginning to shed light on the distinctive roles of RPPs in archaeal RNase P catalysis and the functional coordination among its subunits.

Keywords: RNase P, Single turnover, cooperativity