DNA supercoiling response of the sigma 54-dependent Klebsiella pneumoniae nifL promoter in vitro.
Transcription from the sigma 54-dependent Klebsiella pneumoniae nifL and glnAp2 promoters is activated by the general nitrogen regulatory protein NTRC. Unlike the glnAp2 promoter, which is relatively insensitive to changes in DNA supercoiling, transcription from nifL in vitro in a chloride-based buffer is supercoiling-dependent at physiological salt concentrations. The replacement of chloride with an acetate-based buffer decreases the stringency of the nifL supercoiling response, but open complexes formed on linear nifL promoter DNA under these conditions are unstable and less extensive than those found on supercoiled (form I) DNA. We have introduced mutations in particular elements of the nifL promoter that increase its homology to glnAp2. At the wild-type nifL promoter, sigma 54-RNA polymerase makes only limited contacts with the promoter in the absence of NTRC. However, a G to T change at -26 (nifL74) allows the formation of a stable closed complex with sigma 54-holoenzyme on both linear and form I templates in the absence of the activator. The combination of C to T mutations at -3 and -1 (nifL18) increases the A+T rich nature of the melted region and stabilizes open complexes formed on linear DNA. Open complex formation as a function of superhelical density was assessed at each promoter. Formation of open complexes at glnAp2 peaks at -0.024 and declines at higher superhelical densities, whereas at the wild-type nifL promoter, open complex formation peaks at -0.067 and is not detectable at superhelical densities less than -0.032. Both the nifL74 and nifL18 mutations altered the supercoiling response, increasing the ability to form open complexes at low superhelical densities. The presence of the nifL74 and nifL18 mutations in combination further altered the response of the promoter to DNA supercoiling. These observations suggest that the promoter as a whole, and not any one promoter element, mediates the transcriptional response to DNA supercoiling.
Whitehall
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S
AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
Austin
S
S
Dixon
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R
D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D007711:Klebsiella pneumoniae / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases