Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities.
CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA-binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild-type CYP1 target sites. In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1-(1-200)-peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding. (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1-(1-200)-peptide is usually a monomer in solution but binds DNA as a dimer. Finally, we found evidence for the presence of two half-sites with different measured affinities in the asymmetric sequences of some CYP1 targets.
Näit-Kaoudjt
R
R
Centre de Génétique Moléculaire, Laboratoire propre du Centre Nationalde la Recherche Scientifique associé à l'Universite Pierre etMarie Curie, Gif-sur-Yvette, France.
Williams
R
R
Guiard
B
B
Gervais
M
M
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors