title: The role of rigidity in DNA looping-unlooping by AraC.
abstract: We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.
authors.affiliation: Biology Department, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: T
authors.initials: T
authors.lastname: Harmer
authors.forename: M
authors.initials: M
authors.lastname: Wu
authors.forename: R
authors.initials: R
authors.lastname: Schleif
mesh_terms: D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001089:Arabinose / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000187:drug effects* / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000187:drug effects* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins; D015694:Gene Products, rev / Q000737:chemistry; D015498:HIV-2 / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D009876:Operon; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors; D054321:rev Gene Products, Human Immunodeficiency Virus
chemical_list: D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015694:Gene Products, rev; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D054321:rev Gene Products, Human Immunodeficiency Virus; D004247:DNA; D001089:Arabinose