Biochemical Studies On Methanobrevibacter smithii RNase P - A Model For Mesophilic Type A Archaeal RNase P And A Possible Anti-Obesity Target
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Date
2011-06
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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 archaea. Although both types of archaeal RNase P—types A and M, which are similar to bacterial and eukaryotic RNase P, respectively—have been reconstituted, all archaeal type A holoenzymes studied to date are thermophilic in origin and require high temperatures (≥55oC) for optimal pre-tRNA 5'-processing activity. The availability of a mesophilic archaeal RNase P, such as the one from Methanobrevibacter smithii, however, will permit single molecule-FRET and rapid quench flow kinetic studies, thus far not possible due to limitations imposed by the optimal temperature for thermophilic archaeal RNase P activity. 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 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 biochemical characterization of the M. smithii RNase P holoenzyme, cloning, overexpression, and purification of the protein subunits has been successfully achieved, and the reconstituted holoenzyme shows activity at 37oC. Kinetic studies of the M. smithii RNase P holoenzyme are currently in progress, and preliminary data suggest that the M. smithii RNase P does in fact share many characteristics with other archaeal type A RNase P holoenzymes and might prove to be a useful experimental paradigm.
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RNase P, ribozyme, ribonucleoprotein, archaea, Methanobrevibacter smithii