Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.
Mol Cell Biol
; 13(9): 5524-37, 1993 Sep.
Article
em En
| MEDLINE
| ID: mdl-8355698
ABSTRACT
The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
Fatores de Transcrição
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Proteínas Fúngicas
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Transativadores
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Dedos de Zinco
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Proteínas de Homeodomínio
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Proteínas de Ciclo Celular
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Proteínas de Saccharomyces cerevisiae
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Proteínas de Ligação a DNA
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Mol Cell Biol
Ano de publicação:
1993
Tipo de documento:
Article