Arm-domain interactions in AraC

Abstract

N-terminal deletions extending beyond the sixth amino acid of the Escherichia coli regulator of the l-arabinose operon, AraC, were found to generate constitutive regulatory behavior of the promoter p BAD . Mutagenesis of the DNA coding for the first 20 amino acids of the protein and screening for constitutives yielded mutants across the region whereas screening for mutants that cannot induce p BAD , even in the presence of arabinose, yielded none. These results indicate that the N-terminal arm is not essential for transcription activation, but that it plays an important and active role in holding the system in a non-activating state. Despite the fact that arabinose binds to the N-terminal domain of AraC, mutations were found in the C-terminal domain that weaken the binding of arabinose to the protein. The effects of the mutations could be suppressed by specific mutation in the N-terminal arm or by deletion of the arm. These results, in conjunction with the crystal structures of the N-terminal domain determined in the presence and absence of arabinose, indicate that in the absence of arabinose, the N-terminal arms of the protein bind to the C-terminal DNA binding domains to hold them in a state where the protein prefers to loop. When arabinose is added, the arms are pulled off the C-terminal domains, thereby releasing them to bind to adjacently located DNA half-sites and activate transcription.