title: Tethered particle motion reveals that LacI·DNA loops coexist with a competitor-resistant but apparently unlooped conformation.
abstract: The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.
authors: Department of Physics, University of Michigan, Ann Arbor, Michigan.
authors: Joel D
authors: JD
authors: Revalee
authors: Debye Institute, Molecular Biophysics, Utrecht University, Utrecht, The Netherlands.
authors: Gerhard A
authors: GA
authors: Blab
authors: LSA Biophysics, University of Michigan, Ann Arbor, Michigan.
authors: Henry D
authors: HD
authors: Wilson
authors: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
authors: Jason D
authors: JD
authors: Kahn
authors: Department of Physics, University of Michigan, Ann Arbor, Michigan; LSA Biophysics, University of Michigan, Ann Arbor, Michigan. Electronic address: meiners@umich.edu.
authors: Jens-Christian
authors: JC
authors: Meiners
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D056968:Lac Repressors / Q000737:chemistry / Q000378:metabolism*; D009038:Motion; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D004247:DNA