Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae.

Jensen, L T; Posewitz, M C; Srinivasan, C; Winge, D R

Jensen, L T; Posewitz, M C; Srinivasan, C; Winge, D R.

Afiliación

Jensen LT; Departments of Medicine and Biochemistry, University of Utah Health Science Center, Salt Lake City, Utah 84132, USA.

J Biol Chem ; 273(37): 23805-11, 1998 Sep 11.

Article en En | MEDLINE | ID: mdl-9726991

RESUMEN

Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA. The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex. The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys11 --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function. Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.

Asunto(s)

Cobre/metabolismo; Proteínas Nucleares/metabolismo; Proteínas de Saccharomyces cerevisiae; Saccharomyces cerevisiae/metabolismo; Factores de Transcripción/metabolismo; Sustitución de Aminoácidos; Secuencia de Bases; Sitios de Unión; Clonación Molecular; Cisteína; Escherichia coli; Proteínas Fúngicas/química; Proteínas Fúngicas/genética; Proteínas Fúngicas/metabolismo; Prueba de Complementación Genética; Mutagénesis Sitio-Dirigida; Proteínas Nucleares/química; Proteínas Nucleares/genética; Oligodesoxirribonucleótidos; Sondas de Oligonucleótidos; Regiones Promotoras Genéticas; Proteínas Recombinantes/química; Proteínas Recombinantes/metabolismo; Secuencias Repetitivas de Ácidos Nucleicos; Saccharomyces cerevisiae/genética; Saccharomyces cerevisiae/crecimiento & desarrollo; Factores de Transcripción/química; Factores de Transcripción/genética; Tirosina; Zinc/metabolismo

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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Saccharomyces cerevisiae / Factores de Transcripción / Proteínas Nucleares / Cobre / Proteínas de Saccharomyces cerevisiae Idioma: En Revista: J Biol Chem Año: 1998 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

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