A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity.
We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.
Condee
C W
CW
Department of Microbiology, University of Georgia, Athens 30605.
Summers
A O
AO
D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008628:Mercury / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D008156:Luciferases; D008628:Mercury