The yeast homeodomain protein MATalpha2 shows extended DNA binding specificity in complex with Mcm1.
The MATalpha2 (alpha2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural alpha2-Mcm1 sites with an alpha2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of alpha2 in complex with Mcm1, symmetric substitutions at each position in the alpha2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of alpha2, we performed in vitro alpha2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha2 is extended to these positions.
Zhong
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Waksman Institute, Rutgers University, Piscataway, New Jersey 08855-0759, USA.
Vershon
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D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D018398:Homeodomain Proteins / Q000378:metabolism*; D026341:Minichromosome Maintenance 1 Protein; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006023:Glycoproteins; D018398:Homeodomain Proteins; C062928:MATA2 protein, S cerevisiae; D026341:Minichromosome Maintenance 1 Protein; D012097:Repressor Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA