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J Mol Biol. 2011 Feb 4;405(5):1121-7. doi: 10.1016/j.jmb.2010.11.056. Epub 2010 Dec 7.

Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P.

Author information

1
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.

PMID:
21144851
PMCID:
PMC3025773
DOI:
10.1016/j.jmb.2010.11.056
[Indexed for MEDLINE]
Free PMC Article

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