Poster abstracts

Poster number 11 submitted by I-Ming Cho

Exploring the potential of using archaeal RNase P and external guide sequences for disrupting gene expression

I-Ming Cho (Department of Molecular Genetics, The Ohio State Univertsity), Venkat Gopalan (Department of Biochemistry, The Ohio State Univertsity)

Abstract:
RNase P is an essential endoribonuclease that removes the 5'leader sequence of precursor tRNAs (ptRNAs) during tRNA biogenesis. It functions as a ribonucleoprotein in all three domains of life. Previous studies have shown that in addition to ptRNAs, bacterial, plant, and human RNase P can also cleave model substrates built from two RNAs. For instance, an RNA molecule, called external guide sequence (EGS), could be designed to hybridize with a target RNA to form a bipartite ptRNA-like molecule that is recognized and cleaved by RNase P. Such an approach has been used to cleave and inactivate cellular target RNAs by artificially expressing the desired EGSs inside a cell. Since there is no reliable knock-down procedure in archaea, we are examining the feasibility of using archaeal RNase P for degrading cellular RNAs and thereby down-regulating their function. In vitro characterization of EGS-targeted RNA cleavage was performed using Pyrococcus furiosus (Pfu) RNase P reconstituted from the single RNA subunit, generated by in vitro transcription, and the four known protein subunits, which were co-overexpressed in Escherichia coli and co-purified as two binary complexes under native conditions. We determined that bipartite molecules, comprised of a target RNA substrate and EGS, are indeed efficiently cleaved by RNase P and that EGSs are recycled effectively. We are now attempting to elucidate the underlying basis for EGS recycling. Overall, these studies support the idea of using archaeal RNase P as a tool to knock-down gene expression in archaea in vivo.

Keywords: RNase P, External Guide Sequence, gene expression