Poster abstracts

Poster number 72 submitted by Stella Lai

Towards a molecular model of the Pyrococcus furiosus RNase P holoenzyme

Stella M. Lai (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), Dileep Pulukkunat, Yiren Xu (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Jamie Nickels, Ian Smith (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)

Abstract:
RNase P is an essential enzyme that catalyzes the 5’-maturation of precursor tRNAs (pre-tRNAs) in a Mg²⁺-dependent reaction. While a proteinaceous RNase P is found in several eukaryotes, ribonucleoprotein (RNP) variants have been found in all three domains of life. The RNP forms consist of a catalytic RNA (RPR) and a variable number of protein subunits (RPPs): one in Bacteria, ≤ 5 in Archaea, and ≤ 10 in Eukarya. While the RPR alone is catalytically active at high Mg²⁺ concentrations, addition of the protein cofactor enables the RPR to function at near-physiological conditions by aiding substrate recognition and active site metal ion affinity, as best exemplified by studies on bacterial RNase P. We reported thematic parallels in archaeal RNase P, composed of an RPR + two RPP heterodimers and a lone RPP (POP5-RPP30, RPP21-RPP29 and L7Ae). Addition of POP5-RPP30 increases the RPR’s rate of pre-tRNA cleavage by 60-fold, while RPP21-RPP29 enhances the RPR’s substrate affinity by 15-fold (1). However, little is known about how these archaeal RPPs assemble with the RPR to accomplish their functional roles. We have employed a site-directed hydroxyl-radical footprinting strategy to map the binding sites of each RPP on the Pyrococcus furiosus (Pfu) RPR and a pre-tRNA, building on earlier studies with Escherichia coli RNase P (2) and recent mapping of Pfu L7Ae on the Pfu RPR (unpublished). Here, we constructed single Cys-substituted mutant derivatives of POP5 and RPP29 in which residues at or near their proposed substrate- and RPR-binding interfaces, respectively, were modified to Cys. These mutant derivatives were then purified and modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide (EPD-Fe) to form site-specific protein-tethered nucleases. Reconstituted holoenzymes (RPR + 5 RPPs, including modified POP5 or RPP29) in the absence or presence of pre-tRNA were incubated with hydrogen peroxide and ascorbate to promote OH·-mediated cleavages at proximal sites (< 10 Å) on either the Pfu RPR or the pre-tRNA substrate, which were then mapped. The intra-molecular RPP-RPR and RPP-substrate distance constraints from these ongoing studies are expected to lead to a molecular model of the holoenzyme and provide insights into protein-aided RNA catalysis in archaeal RNase P.

References:
(1) Chen WY, Pulukkunat D, Cho IM, Tsai HY, Gopalan V. (2010) Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex. Nucleic Acids Res. 38: 8316-8327.
(2) Biswas R, Ledman D, Fox RO, Altman S, Gopalan V. (2000) Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. J Mol Biol. 296: 19-31.

Keywords: archaeal RNase P, POP5 and RPP29, hydroxyl-radical footprinting