A plasmid to visualize and assay termination and antitermination of transcription in Escherichia coli

Abstract

To facilitate the analysis of termination and antitermination of transcription in prokaryotes, a complex operon has been assembled into the pBR322 replicon, drawing upon natural and synthetic DNA elements. This operon is initiated from a strongly inducible promoter without temperature restraints. It includes a severe transcription terminator and therefore requires antitermination of transcription to express a downstream lacZ reporter gene. Antitermination can be provided by an upstream N-utilization site from phage lambda, working in conjunction with N protein supplied in trans from a compatible plasmid. In this situation, the nusA gene of Salmonella, substituted into the Escherichia coli host, prevents lacZ function, confirming that a good facsimile of lambda's specific antitermination mechanism has been recreated. The nonessential, easily assayed product of this operon, beta-galactosidase, is also screenable by colony color on chromogenic substrate. The plasmid described will therefore serve as a tester for mutations affecting the various aspects of transcription regulation by termination.