Genetic screens to identify bacterial sRNA regulators.
Small regulatory RNAs (sRNAs) are versatile regulators that have been shown to be involved in the gene regulation of a growing number of biological pathways in bacteria. While finding the targets of a given sRNA has been the focus of many studies, fewer methods have been described to uncover which, if any, sRNAs regulate a given gene. Here I present two genetic screens that are designed to search for sRNAs regulating a gene of interest. Before the screens are performed, a translational fusion is made between the gene of interest and lacZ, designed so that mostly post-transcriptional effects on the gene's expression can be analyzed. I describe here a simple and rapid way to obtain this fusion, even when the transcriptional start site is unknown, by combining PCR or 5'RACE with recombination in the chromosome of a special strain of Escherichia coli. The first genetic screen uses a genomic multicopy library to find regulator genes that, when overexpressed, affect the expression of the fusion. While this technique is a classical genetic screen, particular attention is paid to how it can be used to specifically find sRNAs. A second screen is described that takes advantage of a specific library of sRNAs of E. coli that provides an easier and more rapid way to look for sRNA regulation. The library is transformed into the fusion containing strain using a serial transformation protocol developed in microtiter plates. The transformants can then be directly assayed for effects on the beta-galactosidase activity of the fusion in liquid, providing a precise and rapid way to evaluate sRNA regulation. Use of one or both of these screens should help uncover new pathways of regulation by sRNAs.
Laboratoire de Chimie Bactérienne, Aix-Marseille Université, Institut de Microbiologie de la Méditéranée, Marseille, Cedex 20, France. pmandin@ifr88.cnrs-mrs.fr
Pierre
P
Mandin
D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005821:Genetic Techniques; D007763:Lac Operon / Q000235:genetics; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*
D012329:RNA, Bacterial; D038621:Regulatory Sequences, Ribonucleic Acid