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A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two‐component system
Author(s) -
Mandin Pierre,
Gottesman Susan
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2009.06665.x
Subject(s) - biology , operon , gene , genetics , rna , lac operon , translational regulation , small rna , translation (biology) , regulation of gene expression , gene expression , mutant , messenger rna
Summary 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 β‐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.