A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression.
A series of mutations in the homeo domain of the yeast alpha 2 protein were constructed to test, both in vivo and in vitro, predictions based on the alpha 2-DNA cocrystal structure described by Wolberger et al. (1991). The effects of the mutations were observed in three different contexts using authentic target DNA sequences: alpha 2 binding alone to specific DNA, alpha 2 binding cooperatively with MCM1 to specific DNA, and alpha 2 binding cooperatively with a1 to specific DNA. As expected, changes in the amino acid residues that contact DNA in the X-ray structure severely compromised the ability of alpha 2 to bind DNA alone and to bind DNA cooperatively with MCM1. In contrast, many of these same mutations, including a triple change that altered all the "recognition" residues of helix 3, had little or no effect on the cooperative binding of alpha 2 and a1 to specific DNA, as determined both in vivo and in vitro. These results show that the ability of a homeo domain protein to correctly select and repress target genes does not necessarily depend on the residues commonly implicated in sequence-specific DNA binding.
Waksman Institute, Rutgers University, Piscataway, New Jersey 08855.
A K
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Vershon
Y
Y
Jin
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Johnson
D000595:Amino Acid Sequence; D001483:Base Sequence; D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D005800:Genes, Fungal / Q000235:genetics; D018398:Homeodomain Proteins; D026341:Minichromosome Maintenance 1 Protein; D008954:Models, Biological; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics; D017354:Point Mutation / Q000502:physiology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D013172:Spores, Fungal / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; D026341:Minichromosome Maintenance 1 Protein; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors