Silencing of the Escherichia coli bgl promoter: effects of template supercoiling and cell extracts on promoter activity in vitro.

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RESUMO

Regulation of the Escherichia coli bgl promoter involves the catabolite gene activator protein (CAP) and silencer elements that are located upstream and downstream of the promoter and its CAP binding site. The promoter is kept in a repressed state by the silencer elements and other normally active CAP-dependent or -independent promoters are repressed as well when flanked by these elements. To assess the mechanism of promoter repression, single round in vitro transcription was carried out with plasmids bearing either the wild-type bgl promoter or one of two derivatives that escape repression in vivo by different mechanisms: C234 by improving the CAP binding site of the promoter and delta1 by a deletion within the upstream silencer sequence. Repression of the bgl promoter in vitro was shown to depend on template topology and the presence of cellular factors. With negatively supercoiled templates, all three promoters are transcribed to similar extents by purified E. coli RNA polymerase and no CAP dependence is apparent; with relaxed templates, transcription is CAP dependent, but the levels of transcription of the three promoters are comparable. Addition of crude cell extract to the simple transcription system leads to repression of all three promoter alleles in the absence of CAP. Repression of the mutant alleles but not of the wild-type promoter is completely relieved in the presence of the CAP-cAMP complex. The topology of the DNA template is also important in the differential regulation of these promoters. In the case of C234, repression by cell extract is completely relieved by CAP-cAMP on relaxed or negatively supercoiled templates, while complete derepression of delta1 by CAP-cAMP occurs on negatively supercoiled templates only. Repression by cell extract requires the presence of the histone-like protein H-NS. However, H-NS alone does not appear to be sufficient for specific silencing of the wild-type promoter, since repression of all three promoter alleles caused by purified H-NS protein is completely relieved by the CAP-cAMP complex. These data suggest that template topology, H-NS and other cellular factors are involved in the formation of a specific nucleoprotein structure in the bgl promoter-silencer region; the formation of this nucleoprotein structure keeps an otherwise active promoter in an inactive state.

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