Genetic Screens to Identify Bacterial sRNA Regulators

Abstract

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.