Poster abstracts

Poster number 77 submitted by Emily Wong

Methanobrevivacter smithii RNase P - a model for archaeal mesophilic type A RNase P and a possible anti-obesity target

Emily Wong (Department of Biochemistry, the Ohio State University), I Ming Cho (Department of Molecular Genetics, the Ohio State University), Lien B. Lai (Department of Biochemistry, the Ohio State University), Venkat Gopalan (Departments of Biochemistry and Molecular Genetics, and Center for RNA Biology, the Ohio State University)

Abstract:
RNase P, a universal and essential catalytic ribonucleoprotein responsible for cleaving the 5'-leader of precursor tRNA transcripts, has been successfully reconstituted from several different archaeal representatives. Although both types of archaeal RNase P—types A and M, which are similar to bacterial and eukaryal RNase P, respectively—have been reconstituted, all archaeal type A holoenzymes studied to date are thermophilic in origin and require high temperatures (> 55 ^oC) for optimal pre-tRNA processing activity. The availability of a mesophilic archaeal RNase P (such as the one from Methanobrevibacter smithii) will permit single molecule FRET and rapid quench flow kinetic studies, thus far not possible due to the technical limitations imposed by the optimal temperature for functioning of the thermophilic archaeal RNase P variants. Other payoffs from studies on M. smithii RNase P are likely. Since M. smithii, the predominant archaeon in the human gut, is a methanogen whose syntrophic relationship with saccharolytic bacteria permits increased fermentation of sugars in the digestive process and ultimately a greater caloric absorption from food, it is viewed as a potential anti-obesity target. As the subunit makeup of archaeal RNase P differs from its eukaryal and bacterial relatives, it might be possible to design a specific inhibitor of M. smithii RNase P without harming the animal host or commensal bacteria. Towards the goal of reconstituting the M. smithii RNase P holoenzyme for biochemical characterization and screening of small-molecule inhibitors, the genes encoding the single RNA and protein subunits have been cloned into expression vectors. We will present data from our ongoing efforts to (i) purify recombinant versions of the individual subunits [subsequent to in vitro transcription of the RNA or overexpression of the proteins in E. coli BL21(DE3)], and (ii) reconstitute a functional holoenzyme in vitro from its constituent subunits.

Keywords: RNase P