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NMR Study of Rpp30, an Archaeal RNase P Protein

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Title: NMR Study of Rpp30, an Archaeal RNase P Protein
Creators: Bohlen, Christopher
Advisor: Foster, Mark
Issue Date: 2006-06
Abstract: RNase P, an essential enzyme, is an exceptionally interesting system for the study of protein-protein and protein-RNA interactions in catalysis. Composed of both RNA and protein subunits, the complex catalyzes the hydrolytic cleavage of the 5’ leader sequence of precursor tRNA, a vital step in tRNA processing. While bacterial RNase P is a true ribozyme with a catalytic RNA subunit, it requires a single associated protein for function in vivo. RNase P from archaeal and eukaryal organisms are also composed of requisite protein and RNA subunits. Coordinating at least nine proteins, eukaryotic RNase P is much more complex than the single-protein bacterial system, although both utilize a single RNA subunit. The archaeal enzyme is of intermediate complexity with at least four protein subunits. The functional roles of the RNA and protein subunits are reasonably well characterized in the bacterial system, but how these roles differ in organisms that use more protein subunits remains a mystery. Archaeal RNase P, then, is clearly a prime target for furthering our understanding of this enzyme in particular and of macromolecular cooperation for catalysis in general. Pfu Rpp30 is the largest protein subunit of RNase P in the archaebacterium Pyrococcus furiosus (Pfu). Here, we show that Rpp30 has been recombinantly expressed and isolated by two different purification methods. NMR spectra of the isolated protein revealed dispersed signals characteristic of a well-folded protein, and optimization of spectral quality and subsequent three-dimensional experiments allowed ~90% of 206 assignable backbone amide resonances. These data were subsequently used to characterize an interaction with Pop5, a protein binding partner of Rpp30. Widespread chemical shift perturbations observed for each protein’s NMR spectrum suggest the formation of a tight complex, and mapping perturbations onto three-dimensional structures of Pop5 and Rpp30 indicated a previously unknown (and since corroborated) binding interface between the two molecules. A detailed model of the Rpp30-Pop5 interface will be useful in designing future experiments to fully understand the functional contribution of RNase P subunit interactions. In addition, having high quality and assigned NMR spectra of Rpp30 opens the door for a detailed investigation of its involvement with other subunits using various NMR techniques. Advisor: Mark P. Foster
Series/Report no.: The Ohio State University. Department of Biochemistry Honors Theses; 2006
Keywords: NMR Spectroscopy
Protein Purification
Protein Interactions
Sponsors: College of Arts and Sciences; Undergraduate Research Scholarship
Ohio State Biochemistry Program; Summer Undergraduate Research Experience
URI: http://hdl.handle.net/1811/6585
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