SUBSTITUTION OF BASIC-AMINO-ACIDS IN THE BASIC REGION STABILIZES DNA-BINDING BY E12 HOMODIMERS

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E 47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 ha s previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrop horetic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein co mplexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 c an increase the intrinsic DNA-binding activity of E12 and stabilize th e binding complexes, thus alleviating the repression from the inhibito ry domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bH LH proteins, which ail possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the my ogenic bHLH proteins may need stronger DNA binding domains.