Premium
A reversed approach for finding small RNAs regulating genes of interest
Author(s) -
Mandin Pierre,
Gottesman Susan
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.846.3
Subject(s) - gene , biology , rna , genetics , small rna , lac operon , translation (biology) , translational regulation , post transcriptional regulation , regulation of gene expression , reporter gene , gene expression , messenger rna
In E. coli , the largest class of small regulatory RNAs (sRNAs) binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific target mRNAs. We developed a new method that allows screening for sRNAs regulating genes of interest, using translational fusions of the 5'extremities of those genes to the lacZ reporter gene. A multicopy plasmid library of the E. coli genome can then be screened to determine if over‐expression of certain sRNAs affects the activity of the fusion. This screening method was first applied to the transcriptional regulators DpiB/A. RybC, a small RNA of previously unknown function was found to negatively regulate the expression of dpiB . Using mutants in the dpiB‐lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrated that RybC acts post‐transcriptionally and directly on the dpiBA mRNA. This approach has now been generalized to probe the post‐transcriptional regulation of sigma factors by small RNAs; initial results suggest a complex network of regulation, supporting the usefulness of this approach and the multiple layers of regulation present in bacteria.