Towards reconstitution of human RNase P protein subunits with human and bacterial RNase P RNAs
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Date
2011-06
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The Ohio State University
Abstract
RNase P is an endoribonuclease that catalyzes the removal of 5'-leaders in precursor tRNAs (pre-tRNAs), and it functions in all three domains of life as a ribonucleoprotein (RNP). The holoenzymes consist of an essential, catalytic RNase P RNA subunit (RPR) and a variable number of RNase P protein (RPP) subunits depending on the source. Bacterial RNase P is comprised of one RPR and one RPP. In contrast, eukaryotic RNase P contains one RPR and up to 10 RPPs. Intermediate in complexity, archaeal RNase P consists of one RPR and up to 5 RPPs. Although archaeal and eukaryal RPRs display pre-tRNA processing without RPPs, their activity is 10^2 to 10^6-fold slower than that of bacterial RPR. Despite similarities in the structure of all RPRs, archaeal and eukaryal RPRs are more dependent on cognate RPPs for biological function compared to their bacterial cousin. Recently, we have successfully reconstituted archaeal RNase P in vitro and begun to dissect the roles of individual subunits. However, the multi-subunit eukaryotic RNase P has proven refractory in this regard and prevented a dissection of the functional interplay between RNA and protein subunits, which is vital to understand protein-aided RNA catalysis in this catalytic RNP. Based on insights from studies on archaeal RNase P, we recently purified several human RPPs as binary complexes (with appropriate RPP partners) to decrease the number of possibilities of RPP additions during human RNase P assembly in vitro. This thesis reports on these ongoing purifications and efforts to initiate reconstitution of the 11-subunit human RNase P. In parallel, we also exploited the availability of purified recombinant human RPPs to investigate if any of them (alone or in combinations) aid bacterial RNase P RNA catalysis. Such an undertaking also gains clinical significance given the successful use of bacterial RNase P-based customized ribozymes for selectively cleaving oncogenic and viral mRNAs in human cells.
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2011 Denman Undergraduate Research Forum Winner,1st Place
Keywords
Heterologous reconstitution of E. coli RPR with human RPPs