The SRY cantilever motif discriminates between sequence- and structure-specific DNA recognition: alanine mutagenesis of an HMG box.
The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.
Weiss
M A
MA
Department of Biochemistry & Molecular Biology and Chemistry, University of Chicago, IL 60637-5419, USA. maweiss@midway.uchicago.edu
Ukiyama
E
E
King
C Y
CY
D000409:Alanine / Q000502:physiology*; D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017510:Protein Folding; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497745:SRY protein, human; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D004247:DNA; D000409:Alanine