Application of Site-directed Metal Chelation Techniques to Structure Determination of Pyrococcus furiosus RNase P
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Biochemistry Honors Theses; 2011
Ribonuclease (RNase) P is a ribonucleoprotein complex that is responsible for cleaving the 5' leader sequence during the maturation of tRNA molecules. Its three-dimensional structure yet unknown, RNase P in human nuclei is thought to contain ten protein subunits and one RNA subunit. In contrast, RNase P in the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) is simpler, with only five protein subunits and one RNA subunit. Though the three-dimensional structure of the archaeal holoenzyme is also unknown, all five protein subunits are homologous to corresponding subunits in human RNase P. Due to its more tractable configuration, yet maintained homology, analysis of Pfu RNase P is attractive as a model to provide insights into the structure and function of the human enzyme. The long-term goal of this project is to develop an atomic-resolution model of the Pfu RNase P holoenzyme. To aid in structure determination of this ribonucleoprotein complex, the attachment of an ethylenediaminetetraacetic acid (EDTA) moiety to strategically-located cysteine residues on one of the protein subunits was explored in order to exploit the EDTA moiety’s metal-chelating capabilities in two distinct ways. The chelation of paramagnetic Mn2+ allowed for the execution of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) experiments. This unconventional NMR method enables distance restraints to be obtained over larger distances than conventional NMR methods. The chelation of iron atoms allowed for the implementation of RNA footprinting experiments by utilizing Fe•EDTA-generated hydroxyl radicals: the hydroxyl radicals cleave nucleic acid in a distance-dependent manner. Thus, preparation of chelator-tagged proteins was shown to provide valuable information to constrain structural models of the Pfu RNase P holoenzyme.
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