Arm-domain interactions in AraC.
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 pBAD. 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 pBAD, 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.
Saviola
B
B
Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Seabold
R
R
Schleif
R F
RF
D051777:AraC Transcription Factor; D001089:Arabinose / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009876:Operon; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose