Purification and Biophysical Study of RNase P Proteins 21 and 29 from Methanocaldococcus jannaschii
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Biochemistry Honors Theses; 2010
RNase P is an endonuclease with a rare catalytic RNA core. The enzyme is a ribonucleoprotein (RNP) complex and is primarily responsible for cleaving the 5’ RNA leader sequence from precursor tRNA (ptRNA) molecules. The maturation of tRNA is necessary for all domains of life, which makes RNase P an indispensable and ubiquitous enzyme. This historical prevalence renders RNase P as a potential marker for evolution events. Early life forms like bacteria have very few RNase P Protein (RPP) cofactors; whereas later organisms like eukaryotes have been documented as having more than 10 different protein cofactors. The increasing protein content has subsidized, diminishing RNase P RNA (RPR) content. Ongoing studies of RNase P aim to demonstrate how proteins have assumed biological responsibility from nucleic acids. Our research focuses on the Archaeon Methanocaldococcus jannaschii (Mja). The Mja RNase P has five protein cofactors, two of which are RPP21 and RPP29. The conserved RPP21-RPP29 heterodimer has been implicated in substrate recognition and binding events; however, the latter observation has yet to be applied to Eurkaryotes. Mja was chosen for our study because the RPR is very similar to eukaryotic RPR, and may help bridge the evolutionary gaps between bacteria and humans. Using NMR spectroscopy, our objective is to deduce a solution structure of the RPP21-RPP29 heterodimer. Our efforts have only extended so far as to express and purify both proteins and acquire NMR spectra for RPP29. I have been able to make predictions for the RPP29 secondary structure and have just begun to characterize protein binding; quaternary structure can't yet be determined. These data are a springboard for continuing research that will contribute to the discussion of evolution and protein-protein and protein-RNA interactions.
National Institute of Health