Poster abstracts

Poster number 10 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 (Departments of Biochemistry and Molecular Cellular Developmental Biology Program, The Ohio State University), Yiren Xu (Departments of Biochemistry, The Ohio State University), I-Ming Cho (Departments of Biochemistry and Molecular Genetics, The Ohio State University), Mark P. Foster (Departments of Biochemistry and Ohio State Biochemistry Program, The Ohio State University), Venkat Gopalan (Departments of Biochemistry, Molecular Genetics, and Molecular Cellular Developmental Biology Program, Ohio State Biochemistry Program, The Ohio State University)

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 were computationally identified on the basis of homology 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 measurements. The maximal rate of ptRNA cleavage by the holoenzyme (i.e., RPR + 4RPPs) is ~300-fold faster (and at lower [Mg²⁺]) than that for the catalytic RPR alone. Using functional binary RPP complexes, we established that the RPR’s maximal k_obs increases ~50-fold by addition of POP5-RPP30 but not at all by RPP21-RPP29, though the latter does enhance the k_obs of RPR + POP5-RPP30 by 6-fold. Our data suggest that while POP5-RPP30 directly aids the phosphodiester bond-breaking step, the effects of RPP21-RPP29 manifest only when enabled by POP5-RPP30. Moreover, 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: Ribonuclease P, RNase P protein (RPP), single-turnover