Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P |
| |
Authors: | Cho I-Ming Kazakov Sergei A Gopalan Venkat |
| |
Institution: | Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA. |
| |
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. |
| |
Keywords: | |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|