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Site-directed mutagenesis was conducted in the regulatory region of theEscherichia coli udp gene at promoter sites responsible for binding regulatory proteins CRP and CytR as well as RNA polymerase (the core-promoter containing the –10 sequence). In mutants with an “improved” –10 region, a partial relief from the control of the cAMP–CRP transcription activation complex occurred, and the negative CytR repressor regulation was reduced. In contrast, mutant promoters with a weak Pribnow box or with a deletion that completely eliminates the core-promoter exhibited an increased ability to titrate the CytR protein in vivo. On the other hand, the affinity of CytR for DNA in mutants with an altered –10 region was the same as in the wild-type udp promoter. After introduction of mutations affecting binding sites for CRP (CRP1 and CRP2), the negative effect of the CytR protein on promoter transcription was fully abolished. The CRP1 binding site was shown to play the main role in the activation of the promoter by the cAMP–CRP complex, whereas the CRP2 site participates in the formation of the repressor complex. Mutations in the main and additional CytR binding sites were isolated and characterized. On the basis of these data, it is concluded that the modification of each structural element of the udp regulatory region (binding sites for CytR, CRP, or RNA polymerase) caused changes in the overall pattern of the promoter regulation. |
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