COVALENTLY LINKING BHLH SUBUNITS OF MASH-1 INCREASES SPECIFICITY OF DNA-BINDING

MASH-1, a member of the basic-helix-loop-helix (BHLH) family of transc ription factors, promotes the differentiation of committed neuronal pr ecursor cells. In vitro, MASH-I displays only marginal DNA sequence sp ecificity. We have produced a MASH-I variant, MASH-GGC, by introducing the tripeptide Gly-Gly-Cys at the C-terminal end of the BHLH domain. Under reducing conditions the properties of MASH-GGC and of the BHLH d omain of MASH-1 were very similar. Like MASH-1, reduced MASH-GGC showe d little specificity of DNA binding, CD spectroscopy revealed that bot h proteins underwent a conformational change from a largely unfolded t o a mainly cc-helical conformation upon binding to DNA. When the subun its of MASH-GGC were linked through a disulfide bond, the folded confo rmation was stable over a wide concentration range (2.5 nM to 2 mu M) even in the absence of DNA. Oxidized MASH-GGC bound to E-box-containin g sequences half-maximally at 148 nM, compared to 458 nM for the reduc ed form. Therefore, even when the change from a monomeric to a dimeric species was taken into account, the affinity for E-box-containing DNA sequences was increased. Surprisingly, the apparent dissociation cons tant for the complex with DNA not containing E-box sequences was incre ased upon oxidation. Therefore, despite the large distance between the disulfide bridge and the protein-DNA interface, covalently linking th e subunits of MASH-I increased the specificity of DNA binding signific antly. In vivo, such an increase of the intrinsic DNA binding specific ity might be achieved through interactions with other proteins of the transcriptional machinery.