RESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas de Homeodomínio , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/isolamento & purificação , Fatores de Transcrição/metabolismo , Dedos de Zinco , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Homeodomínio , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Transcrição Gênica , Dedos de Zinco , Sequência de Bases , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-Atividade , Transativadores/metabolismoRESUMO
SWI5 encodes a zinc-finger protein required for expression of the yeast HO gene. Using Swi5 protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the Swi5 stimulatory factor as the product of the GRF10 gene, which encodes a yeast homeodomain protein. GRF10, also known as PHO2 and BAS2, is a transcriptional activator of the PHO5 acid phosphatase gene and the HIS4 histidine biosynthesis gene. Grf10 protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disassociation rates indicates that the Grf10-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or Grf10. Finally, we show that HO expression is reduced in yeast strains containing grf10 null mutations and that full expression of a heterologous promoter containing a SWI5-dependent HO upstream activation sequence element requires GRF10.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Homeodomínio , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Desoxirribonucleoproteínas/química , Genes Homeobox , Substâncias Macromoleculares , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Dedos de ZincoRESUMO
SW15 encodes a zinc finger DNA binding protein required for the transcription of the Saccharomyces cerevisiae HO gene, and PHO2 encodes a homeodomain DNA binding protein. In vitro biochemical studies using purified Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p bind cooperatively to the HO promoter. In this report we investigate the regions of the Swi5p and Pho2p proteins required for cooperative DNA binding. The analysis of each protein gives a similar result: the zinc finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are required for cooperative DNA binding. In vitro and in vivo experiments were conducted with promoters with altered spacing between the Pho2p and Swi5p binding sites. Mutations that increased the distance between the two binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity. These observations suggest that the interaction domains of Swi5p and Pho2p are flexible and can tolerate an increase in distance between the two binding sites. The mechanism of the cooperative DNA binding by Swi5p and Pho2p is discussed.