Characterization of the DNA binding and bending HMG domain of the yeast hypoxic repressor Rox1.
The yeast Rox1 hypoxic transcriptional repressor protein binds to and bends a specific DNA sequence through an HMG domain located at the N-terminus. To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains. The mutants were characterized in terms of their ability to repress the hypoxic gene ANB1 and the auto-repressed ROX1 gene in vivo. The mutant HMG domains were fused to maltose binding protein and expressed in and purified from Escherichia coli and their relative affinities for DNA and ability to bend DNA were determined. A model of the structure of the Rox1 HMG domain was derived using sequence similarities between Rox1 and the human protein SRY, the structure of which has been determined. The results of the mutational analysis are interpreted in terms of the model structure of Rox1.
Deckert
J
J
Department of Biological Sciences, University at Albany/SUNY, Albany, NY 12222, USA.
Khalaf
R A
RA
Hwang
S M
SM
Zitomer
R S
RS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C083348:ROX1 protein, S cerevisiae; D012097:Repressor Proteins; C497745:SRY protein, human; D029701:Saccharomyces cerevisiae Proteins; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D004247:DNA