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J Biol Chem. 2009 Sep 25;284(39):26394-401. doi: 10.1074/jbc.M109.015875. Epub 2009 Jul 28.

Bacillus subtilis trp Leader RNA: RNase J1 endonuclease cleavage specificity and PNPase processing.

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1
Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of New York University, New York, New York 10029-6574, USA.

Abstract

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3' transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3'-to-5' exonuclease processing from the native 3' end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5' tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5' end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.

PMID:
19638340
PMCID:
PMC2785327
DOI:
10.1074/jbc.M109.015875
[Indexed for MEDLINE]
Free PMC Article
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