[Involvement of sigma S and sigma 70 subunits of RNA polymerase and the CRP protein in the regulation of microcin C51 operon expression]. / Uchastie sigmy S, sigmy 70 sub''edinits RNK-polimerazy i belka CRP v reguliatsii ékspressii operona mikrotsina C51.

Expression of the microcin C51 operon in Escherichia coli cells is activated during cell entry into the stationary growth phase and depends on the sigmaS subunit of RNA polymerase (RpoS). The null rpoS mutations retained the residual expression level of the transcriptional P(mcc)-lac fusion, which indicates that other sigma subunit can participate in the regulation of transcription of the microcin C51 operon. Data presented in this work show that the overproduction of sigma70 in rpoS- cells diminished the level of P(mcc)-lac expression, as in wild-type cells, which seems to be the consequence of competition between sigma factors for a limited number of core RNA polymerase molecules. In the presence of the rpoD800 mutation that renders sigma70 temperature-sensitive, expression of P(mcc)-lac was not induced in the phase of delayed culture growth at nonpermissive temperature, which indicates that sigma70 is indispensable for microcin operon expression. Point substitutions in the -10 P(mcc) region, leading to the formation of 5'-TGaTATAAT-3' site, enhanced promoter activity but did not affect the relationship between P(mcc)-lac transcription and growth phase, sigmaS, and the activator protein CRP. The activator protein CRP was shown to bind a DNA fragment containing the TGTGA(AATGAA)TCTAT site in the -59.5 bp position relative to the start site of transcription. Mutation in the ssrI gene encoding 6S RNA did not disturb P9mcc)-lac expression; these results indicate that 6S RNA does not participate in the regulation of microcin C51 operon expression.

[Functional interrelationship between elements of the Escherichia coli udp gene promotor responsible for binding regulatory proteins CytR, CRP, and RNA polymerase]. / Funktsional'noe perekryvanie élementov promotora gena udp Escherichia coli, otvetstvennykh za sviazyvanie reguliatornykh belkov CytR, CRP i RNK-polimerazy.

Site-directed mutagenesis was conducted in the regulatory region of the Escherichia 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 block 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.