How AraC interacts specifically with its target DNAs.
Previous work indicates that one subunit of the AraC protein dimer binds to a DNA target araI1, of 17 base-pairs. We systematically substituted every base-pair in a synthetic araI1 target with the three possible alternatives and then tested binding of araI1 and of these 51 DNA targets to AraC by quantitative gel shift analysis in the presence of L-arabinose. We found that every substitution of the underlined bases reduces AraC binding tenfold or more: 5' TAGCATTTTTATCCATA 3'. Substitutions at other bases have little or no effect. In the absence of L-arabinose we observed a sixfold reduction of binding of AraC to araI1. We have designated the 5' AGC sequence the A-box and the 5'TCCATA sequence the B-box. We synthesised DNA targets containing either two A or two B-boxes with the natural araI1-I2 spacing. Wild-type AraC binds both targets in the presence of L-arabinose in a gel shift-experiment. In the absence of L-arabinose, AraC binds only to the double B-box. We then tested various AraC mutant proteins in the same way. S208A and H212A bind to the double B-box but not to the double A-box in the presence or absence of L-arabinose. D256A binds to the double A-box, but not to the double B-box, in the presence of L-arabinose but not in its absence. The implications of these results for the mechanism of AraC induction by L-arabinose are discussed.
Niland
P
P
Institut für Genetik der Universität zu Köln, Germany.
Hühne
R
R
Müller-Hill
B
B
D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001089:Arabinose / Q000494:pharmacology; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D017354:Point Mutation; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose