A plasmid to visualize and assay termination and antitermination of transcription in Escherichia coli.
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.
Franklin
N C
NC
Biology Department, University of Utah, Salt Lake City 84112.
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005801:Genes, Homeobox; D005809:Genes, Regulator; D005838:Genotype; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012093:Replicon; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic