Central issues surrounding the basic helix-loop-helix (bHLH) superfamily of dimeric transcription factors concern how specificity of partner selection and DNA binding are achieved. bHLH proteins bind DNA through the basic sequence that is contiguous with a helix-loop-helix dimerization domain. For the two subgroups within the family, dimerization is further regulated by an adjacent Per-Arnt-Sim homology (PAS) or leucine zipper (LZ) domain. We provide evidence that for the bHLH⅐PAS transcription

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... factors Dioxin Receptor (DR) and Arnt, the DR PAS A domain has a unique interaction with the bHLH region that underpins both dimerization strength and affinity for an atypical E-box DNA sequence. A PAS swap heterodimer, where the DR bHLH domain was fused to Arnt PAS A and the Arnt bHLH fused to DR PAS A, gave strong DNA binding, but dimerization was only effective with the native arrangement, suggesting the PAS A domain is critical for each process via distinct mechanisms. LZ domains, which regulate heterodimerization for the bHLH⅐LZ family members Myc and Max, could not replace the PAS domains for either dimerization or DNA binding in the DR/Arnt heterodimer. In vitro footprinting revealed that the PAS domains influence the conformation of target DNA in a manner consistent with DNA bending. These results provide the first insights for understanding mechanisms of selective dimerization and DNA interaction that distinguish bHLH⅐PAS proteins from the broader bHLH superfamily. FIGURE 3. The region of DR required for stable dimerization and strong DNA binding. A, the entire and intact bHLH⅐PAS is necessary for dimerization. The native or chimeric proteins indicated were expressed in bacteria, and the material recovered after nickel affinity chromatography (Ni) was purified further by size exclusion chromatography (left panels). Proteins recovered in fractions collected across the elution profile (bar) were analyzed by SDS-PAGE (right panels). The positions of the peaks corresponding to native DR/Arnt DNA Binding and the Dioxin Receptor PAS Domain