Initiation of decay of Bacillus subtilis trp leader RNA

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine of New York University, New York, NY 10029, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2007; 282(28):20238-44. DOI: 10.1074/jbc.M702747200
Source: PubMed


Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3' end of trp leader RNA and PNPase digestion of trp leader RNA from the 3' end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.

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    • "If the initial destabilizing cleavage by RNase Y occurred in the 3 -UTR, the lack of degradation by PNPase might stabilize the cleaved RNA sufficiently to yield a protein product. As in E. coli, PNPase from B. subtilis is sensitive to RNA secondary structure (Deikus and Bechhofer 2007). Bacillus subtilis has at least three more 3 -5 exoribonucleases that help to degrade mRNAs: RNase PH acts by phosphorolysis like PNPase, while RNase R and YhaM are hydrolytic enzymes. "
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    • "We reasoned that, if RNase J1 cleavage at this site was constitutive and was involved in initiating decay, then altering the site such that RNase J1 would no longer recognize it might result in a longer DermC mRNA half-life. A similar sequence change at an RNase J1 target site in trp leader RNA resulted in a fourfold increase in RNA stability (Deikus and Bechhofer 2007). The effect of the GC-rich sequence on RNase J1 recognition was confirmed by in vitro analysis of RNase J1 cleavage of 59-end- labeled DermC mRNA (data not shown). "
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