Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans.

Kulmburg, P; Sequeval, Daria; Lenouvel, François; Mathieu, Martine; Felenbok, Béatrice

doi:10.1128/mcb.12.5.1932

Cited by 57 publications

(60 citation statements)

References 24 publications

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“…chain reaction and cloned in frame with the GST gene in the pGEX-2T plasmid (Pharmacia Biotech Inc.). The protein was expressed from the tac promoter and purified as described earlier (17,14). The protein obtained was 80 -90% pure as judged by SDS gel electrophoresis using Coomassie Blue staining.…”

Section: Methodsmentioning

confidence: 99%

“…Methylation interference was performed as described previously (14). Briefly, single-strand DNA was end-labeled with [␥-32 P]ATP (3000 Ci/ mmol), annealed with its complementary nonlabeled strand, and used for EMSAs.…”

Section: Methodsmentioning

confidence: 99%

“…It contains in its DNA-binding domain, between the third and fourth cysteines, an unusual extended sequence of 16 residues instead of the six to eight usually found, and no predicted dimerization regions were found downstream of the zinc cluster. Furthermore, unlike the other members of this family, AlcR appears to bind with the same affinity to both symmetric and asymmetric sites containing the consensus motif 5Ј-CCGCA-3Ј (14,15). Both types of targets localized in the alcR and alcA promoters have been shown to be functional in vivo (15,16).…”

mentioning

confidence: 99%

“…Furthermore, unlike the other members of this family, AlcR appears to bind with the same affinity to both symmetric and asymmetric sites containing the consensus motif 5Ј-CCGCA-3Ј (14,15). Both types of targets localized in the alcR and alcA promoters have been shown to be functional in vivo (15,16).…”

mentioning

confidence: 99%

“…The AlcR-binding sites have been determined previously using a bacterial expression system with a glutathione S-transferase (GST) 1 fusion protein (GST-AlcR-(7-60)) (14,15). Bacterially expressed fusion proteins are widely used to identify specific DNA sequences that are recognized by regulators.…”

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confidence: 99%

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In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

Lenouvel

Nikolaev

Felenbok

1997

Journal of Biological Chemistry

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AlcR is the transactivator mediating transcriptional induction of the alc gene cluster in Aspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn 2 Cys 6 family by several features. In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A. nidulans. Here, we show that unlike the other proteins of the Zn 2 Cys 6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets. Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats. The consensus core has been determined precisely (5 -CCGCN-3 ), and the AlcR-binding site in the aldA promoter has been localized. The sequence downstream of the zinc cluster is necessary for high affinity binding. Furthermore, our data show that the use of the carrier protein glutathione S-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

Lenouvel

Nikolaev

Felenbok

1997

Journal of Biological Chemistry

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pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene.

et al. 1993

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Transcription of the ipnA gene encoding isopenicillin N synthetase, an enzyme of secondary metabolism, is under the control of the pH regulatory system in the fungus Aspergillus nidulans. External alkaline pH or mutations in pacC, the wide domain regulatory gene which mediates pH regulation, override carbon regulation of ipnA transcript levels, resulting in elevation of the levels of this message in sucrose broth. Strains carrying these mutations, which mimic growth at alkaline pH, produce higher levels of penicillins when grown in sucrose broth compared with the wild type strain grown under carbon derepressing conditions. ipnA transcription is regulated by carbon (C) source, but extreme mutations in creA (the regulatory gene mediating carbon catabolite repression) only slightly increase repressed transcript levels. Precise deletion of the only in vitro CreA binding site present in a region of the ipnA promoter involved in carbon regulation has no effect on ipnA expression. The levels of ipnA transcript in broths with acetate or glycerol as principal C sources are inconsistent with direct or indirect creA‐mediated transcriptional control of the gene. We conclude that a second, creA‐independent mechanism of carbon repression controls expression of this gene. All derepressing C sources tested result in alkalinization of the growth media. In contrast, all repressing C sources result in external acidification. Neither acidic external pH nor pal mutations, mimicking the effects of growth at acid pH, prevent carbon derepression, providing strong support for independent regulatory mechanisms, one mediating carbon regulation (via thus far unidentified genes) and another mediating pH regulation (through the pacC‐encoded transcriptional regulator). External pH measurements suggest that these two independent forms of regulation normally act in concert. We propose that external alkalinity represents a physiological signal which triggers penicillin biosynthesis.

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The Aspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator.

1994

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Carbon catabolite repression in Aspergillus nidulans is mediated by a negative‐acting protein coded by the creA gene. We have investigated how CREA controls the expression of the ethanol regulon genes. CREA is a major component of the control of this regulon. Its presence in the cell results in a permanent, albeit partial, repression of the alc genes under all physiological growth conditions, even when the fungus is grown on carbon sources considered to be non‐repressing. A crucial step in the control processes is the repression of the positive‐acting specific regulatory gene alcR, by the binding of CREA on its cognate target sites on the alcR promoter. The removal of one of these targets, URSA, results in a 50% derepression of the alcR gene. Furthermore, the presence of this sequence contributes directly to the low alcR expression under nonrepressing conditions and reduces alcR promoter function by at least 100‐fold. CREA acts both on the regulatory gene alcR and directly on the two structural genes alcA and aldA, as glucose repression of the latter genes occurs in strains where alcR transcription is driven by a strong constitutive and derepressed promoter. In vivo and in vitro competition experiments show that CREA acts by competing directly with the binding of the ALCR activator for the same region of the alcR promoter, a region which encompasses overlapping targets for both regulatory proteins. These data are consistent with a model in which the activating and repressing regulatory proteins compete to regulate expression of the ethanol regulon genes.

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Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans.

Cited by 57 publications

References 24 publications

In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene.

The Aspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator.

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