Homeodomain-DNA interactions of the Pho2 protein are promoter-dependent.
The homeodomain (HD) is a conserved sequence-specific DNA-binding motif found in many eukaryotic transcriptional regulatory proteins. Despite the wealth of in vitro data on the mechanism HD proteins use to bind DNA, comparatively little is known about the roles of individual residues in these domains in vivo . The Saccharomyces cerevisiae Pho2 protein contains a HD that shares significant sequence identity with the Drosophila Engrailed protein. We have used the co-crystal structure of Engrailed as a model to predict how Pho2 might contact DNA and have examined how individual residues of the Pho2 HD contribute to transcriptional activation in vivo and to DNA binding in vitro. Our results demonstrate that Pho2 and Engrailed share many similar DNA-binding characteristics. However, our results also show that some highly conserved residues, which contact the DNA in many HD structures, make relatively small contributions to the DNA-binding affinity and in vivo activity of the Pho2 protein. We also show that the N-terminal arm of the Pho2 HD is a critical component in determining the DNA-binding specificity of the protein and that the requirements for residues in the N-terminal arm are promoter-dependent for Pho2 transcriptional activation and DNA binding.
Justice
M C
MC
Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855, USA.
Hogan
B P
BP
Vershon
A K
AK
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation
D005656:Fungal Proteins; D018398:Homeodomain Proteins; C051611:PHO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D004247:DNA