title: Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P.
abstract: RNA-mediated RNA cleavage events are being increasingly exploited to disrupt RNA function, an important objective in post-genomic biology. RNase P, a ribonucleoprotein enzyme that catalyzes the removal of 5'-leaders from precursor tRNAs, has previously been utilized for sequence-specific cleavage of cellular RNAs. In one of these strategies, borne out in bacterial and mammalian cell culture, an external guide sequence (EGS) RNA base-paired to a target RNA makes the latter a substrate for endogenous RNase P by rendering the bipartite target RNA-EGS complex a precursor tRNA structural mimic. In this study, we first obtained evidence that four different mesophilic and thermophilic archaeal RNase P holoenzymes, reconstituted in vitro using their respective constituent RNA and protein subunits, recognize and cleave such substrate-EGS complexes. We further demonstrate that these EGSs engage in multiple rounds of substrate recognition while assisting archaeal RNase P-mediated cleavage of a target RNA in vitro. Taken together, the EGS-based approach merits consideration as a gene knockdown tool in archaea.
authors.affiliation: Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: I-Ming
authors.initials: IM
authors.lastname: Cho
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Kazakov
authors.forename: Venkat
authors.initials: V
authors.lastname: Gopalan
mesh_terms: D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry; D019642:RNA, Archaeal / Q000737:chemistry*; D043262:Ribonuclease P / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry; D058727:RNA, Small Untranslated
chemical_list: D012322:RNA Precursors; D019642:RNA, Archaeal; D012261:Ribonucleoproteins; D043262:Ribonuclease P; D058727:RNA, Small Untranslated