Abstract
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.
Highlights
Homeodomain proteins are a family of transcription factors involved in many developmental and cellular processes and have been found in almost every eukaryotic organism [1,2,3,4]
The natural sites are highly conserved, there are variations at some positions that may result in different levels of ␣2-Mcm1-mediated repression. To verify that these ␣2Mcm1 sites are all functional repressor sites and to measure their relative strength for repression in the same promoter context, oligonucleotides containing the sites were inserted into the promoter region of a CYC1-lacZ reporter plasmid, and the level of expression from the promoter was assayed by measuring -galactosidase activity (Fig. 1)
The five natural ␣2-Mcm1 binding sites that have been identified in the promoter regions of asg are highly conserved
Summary
Homeodomain proteins are a family of transcription factors involved in many developmental and cellular processes and have been found in almost every eukaryotic organism [1,2,3,4]. Since many of the studies which examine homeodomain binding sites have been done in the absence of cofactors, this may explain why in some cases sites selected in vitro may not be functional sites for the homeodomain protein complexes in vivo. To address this issue and to understand how homeodomain proteins recognize specific sites, we have investigated the DNA binding specificity of ␣2, a yeast homeodomain protein, in which the natural target sites and cofactors are well known. In complex with Mcm, the sequence specificity of DNA binding by ␣2 is apparently more extended than on its own These results suggest one explanation for DNA recognition sites determined in. Vitro by proteins in the absence of their cofactors may not function as optimal sites in vivo
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