A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two-component system.
In Escherichia coli, the largest class of small regulatory RNAs binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific mRNAs. While recent studies have shown that some mRNAs could be subject to post-transcriptional regulation by sRNAs (e.g. mRNAs found by co-immunoprecipitation with Hfq), no method has yet been described to identify small RNAs that regulate them. We developed a method to easily make translational fusions of genes of interest to the lacZ reporter gene, under the control of a P(BAD)-inducible promoter. A multicopy plasmid library of the E. coli genome can then be used to screen for small RNAs that affect the activity of the fusion. This screening method was first applied to the dpiB gene from the dpiBA operon, which encodes a two-component signal transduction system involved in the SOS response to beta-lactams. One small RNA, RybC, was found to negatively regulate the expression of dpiB. Using mutants in the dpiB-lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrate that RybC directly base pairs with the dpiBA mRNA.
Laboratory of Molecular Biology, National Cancer Institute, Center for Cancer Research, Bethesda, MD 20892, USA.
Pierre
P
Mandin
Susan
S
Gottesman
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013014:SOS Response, Genetics; D016415:Sequence Alignment; D015398:Signal Transduction
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011494:Protein Kinases; C460095:CitA protein, E coli