Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site

Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ah receptor nuclear translocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers an d analyzed the ability of these dimers to interact with their derived conse nsus sequences and activate genes. chi(2) analysis of the selected DNA reco gnition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonst rated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer, These flanking sequences also regu late the ability of ARNT to competitively displace the c-Myc/Max heterodime r from a CACGTG-containing sequence. However, transient transfection analys es in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abiliti es to activate transcription through each other's consensus sequences. Take n together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by fl anking nucleotides, transcriptional activity may also be determined by othe r factors, such as cellular concentrations of these proteins and their co-a ctivators.