Apo-AraC actively seeks to loop.
In the absence of arabinose and interactions with other proteins, AraC, the activator-repressor that regulates the araBAD operon in Escherichia coli, was found to prefer participating in DNA looping interactions between the two well-separated DNA half-sites, araI1 and araO2 at their normal separation of 211 base-pairs rather than binding to these same two half-sites when they are adjacent to one another. On the addition of arabinose, AraC preferred to bind to the adjacently located half-sites. Inverting the distally located araO2 half-site eliminated the looping preference. These results demonstrate that apo-AraC possesses an intrinsic looping preference that is eliminated by the presence of arabinose. We developed a method for the accurate determination of the relative affinities of AraC for the DNA half-sites araI1, araI2, and araO2 and non-specific DNA. These affinities allowed accurate calculation of basal level and induced levels of expression from pBAD under a wide variety of natural and mutant conditions. The calculations independently predicted the looping preference of apo-AraC.
Seabold
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Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Schleif
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RF
D001059:Apoproteins / Q000096:biosynthesis / Q000378:metabolism*; D051777:AraC Transcription Factor; D001089:Arabinose / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008433:Mathematics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D009876:Operon; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000096:biosynthesis / Q000378:metabolism*; D014157:Transcription Factors; D001616:beta-Galactosidase / Q000096:biosynthesis*
D001059:Apoproteins; D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001089:Arabinose; D001616:beta-Galactosidase