title: Structure of HAP1-PC7 bound to DNA: implications for DNA recognition and allosteric effects of DNA-binding on transcriptional activation.
abstract: HAP1 is a transcription factor in yeast whose DNA-binding domain has been implicated in directly affecting transcriptional activation. Two separate mutations in the DNA-binding domain, S63G (HAP1-PC7) and S63R (HAP1-18), retain wild-type binding affinity. However, HAP1-PC7 is transcriptionally silent while HAP1-18 shows highly elevated levels of transcription. We have determined the X-ray crystal structure of the DNA-binding domain of HAP1-PC7 bound to its DNA target, UAS(CYC7), and compared it to the previously solved HAP1-wt and HAP1-18 complexes to UAS(CYC7). Additionally, we have quantitatively compared the DNA-binding affinity and specificity of the HAP1-PC7, HAP1-18 and HAP1-wt DNA-binding domains. We show that, although the DNA-binding domains of these three proteins bind UAS(CYC7) with comparable affinity and specificity, the protein-DNA interactions are dramatically different between the three complexes. Conserved protein-DNA interactions are largely restricted to an internal DNA sequence that excludes one of the two conserved DNA half-sites of UAS(CYC7) suggesting a mode of recognition distinct from other HAP1 family members. Alternative protein-DNA interactions result in divergent DNA configurations between the three complexes. These results suggest that the differential transcriptional activities of the HAP1, HAP1-18 and HAP1-PC7 proteins are due, at least in part, to alternative protein-DNA contacts, and implies that HAP1-DNA interactions have direct allosteric effects on transcriptional activation.
authors.affiliation: The Wistar Institute and The Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Lukens
authors.forename: D A
authors.initials: DA
authors.lastname: King
authors.forename: R
authors.initials: R
authors.lastname: Marmorstein
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D020218:Response Elements / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D015003:Yeasts / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase