title: Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules.
abstract: The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled.
authors.affiliation: Graduate program in Biophysics and Structural Biology, Brandeis University, Waltham, MA, USA.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sanchez
authors.forename: Melisa L
authors.initials: ML
authors.lastname: Osborne
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.forename: Jane
authors.initials: J
authors.lastname: Kondev
authors.forename: Jeff
authors.initials: J
authors.lastname: Gelles
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D007763:Lac Operon; D056968:Lac Repressors / Q000235:genetics; D007785:Lactose / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012321:DNA-Directed RNA Polymerases; D007785:Lactose