The Yeast a1 and α2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding

Abstract

In diploid cells of the yeast Saccharomyces cerevisiae, the alpha2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the alpha2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-alpha2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha2 or a1 protein. Interestingly, an alpha2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of alpha2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the alpha2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.

Life History and Developmental Processes in the BasidiomyceteCoprinus cinereus

“…In the heterodimeric complex, the HD1 homeodomain motif of ␣2 is not essential for function, unlike the HD2 motif of a1 (189,504). Similarly, in C. cinereus, natural and artificial fusions between HD2 and parts of HD1 proteins and mutagenesis of the recognition helices of the HD1 and HD2 motifs revealed that the HD1 but not the HD2 motif can be eliminated without loss of function in activating sexual development and repression of oidiation (13,232,259).…”

Genomic dissection of the cell-type-specification circuit in Saccharomyces cerevisiae

“…For the salt-sensitivity experiment, the a1-␣2 site in the endogenous HOG1 gene promoter was replaced by integration of Kluyveromyces lactis URA3, which was subsequently replaced by the integration of an oligonucleotidegenerated construct to restore the HOG1 promoter with a modified a1-␣2-binding site: GCGTGgCGGATTTTACggCC (lowercase ''g'' replaced T, A, and T in the wild-type sequence). These nucleotides are highly conserved among a1-␣2-binding sites and have been demonstrated to be critical for a1-␣2 binding and repression (10). The promoter was sequenced to verify correct integration.…”

Altering the DNA-binding Specificity of the Yeast Matα2 Homeodomain Protein

“…Plasmids-Transcription reporter plasmids with wild-type or mutant ␣2-Mcm1 or a1-␣2 binding sites were previously constructed by inserting double-stranded oligonucleotides containing these sites with TCGA overhangs into the XhoI site between the UAS and TATA elements of the CYC1-lacZ promoter of pTBA23 (27)(28)(29). The wild-type ␣2-Mcm1 site used in these experiments is a symmetric site derived from a consensus of the wild-type sites found upstream of a-specific genes (27).…”

“…The mutant sites were named by describing the original nucleotide, the positions mutated, and the substituted nucleotide. For instance, T 3 A is a symmetric mutant in which T at position 3 is mutated to A, and A at the symmetric position 28 is mutated to T. The a1-␣2 site used in these studies is derived from a consensus of the wild-type binding sites found upstream of haploidspecific genes (28). Mutant derivatives of the a1-␣2 site contain base pair substitutions only in the ␣2 half-site and are in the same relative position as the mutations in the ␣2-Mcm1 site.…”

“…The ␣2 mutants were expressed from derivatives of the bacterial expression vectors pJM163 (29) and pYJ195 (28), which contain an N-terminal 6-His-tagged MAT␣2 gene coding for the full-length protein or a C-terminal fragment of residues 123-210, respectively. Derivatives of pJM163 containing mutations in ␣2 were constructed by replacing the 490-base pair BglII-NheI fragment of pJM164 with the 570-base pair BglII-NheI fragments from the mutants in pJM130 (29).…”