Detecting RNA Regulatory Interactions in Bacterial Cells

Abstract

Non-coding RNAs are involved in the regulation of most major cellular process in Escherichia coli. With current technologies, many of these molecules have been identified; however, the full scope of their regulatory interactions is still unknown. None of the techniques currently in use employ the regulatory effect of the RNAs, which is the major unifying attribute of these molecules, for their identification. This thesis presents projects involving the design of a dual-reporter plasmid and screening method in the discovery and characterization of RNA regulatory interactions The first project details the engineering of the dual reporter plasmid. This vector utilizes one fluorescent protein to detect regulatory events and a second to normalize for off-target effects. The second project utilizes this tool in the discovery and characterization of novel regulatory responses. This is accomplished by screening a library of intergenic regions for regulatory responses against a collection of metabolite. Interesting interactions involving nitrogen abundance, iron and uracil are identified and further examined. Finally, this thesis examines how this technology can be further expanded for the study of RNA regulatory functions. The use of the screening method for the detection of regulatory events caused by alternative minimal media composition and the potential for the dual reporter plasmid to aid in the study of riboswitches are investigated.