Linkage between oligomerization and DNA binding in Drosophila doublesex proteins.
The doublesex gene of Drosophila melanogaster encodes DSXM protein in males and DSXF protein in females. Dimers of each protein bind a DNA site from which DSXM represses and DSXF activates transcription. Amino acids 1-397 are identical between the proteins and include a domain (DBD) for both DNA binding and protein oligomerization. The remaining nonhomologous and therefore sex-specific C-termini include an essential part of a second oligomerization domain. We have used mobility shift assays to investigate the effects these three oligomerization domains (DBD and two sex-specific) have on DSX dimerization and DNA binding. The intrinsic DNA binding affinities of DSXM and DSXF dimers are indistinguishable from each other (0.17 +/- 0.04 nM) and slightly lower than that of DBD dimers (0.48 nM). In contrast, the dimerization dissociation constants of DSXM (0.05 +/- 0.02 nM) and DSXF (0.16 +/- 0.05 nM) are slightly different, but 4 orders of magnitude lower than that of DBD (430 nM). Thus sequences outside of DBD, presumably the sex-specific oligomerization domains, have substantial effects on apparent DNA binding affinity through thermodynamically linked effects on dimerization of full-length proteins. Further, when two DNA binding sites are adjacent, DBD dimers show no binding cooperativity, whereas full-length dimers bind with 2-fold different cooperativity (DSXF, k12 = 2.6; DSXM, k12 = 5.4). This suggests that the sex-specific domains may have a second effect on DNA binding, namely, an effect on binding cooperativity that depends on the number and arrangement of DNA sites.
The Rosenstiel Center,The Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
S
S
Cho
P C
PC
Wensink
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019281:Dimerization; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005260:Female; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D008297:Male; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012727:Sex Characteristics; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D019476:Insect Proteins; D014157:Transcription Factors; D004247:DNA