title: Gene repression by minimal lac loops in vivo.
abstract: The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Bond
authors.forename: Justin P
authors.initials: JP
authors.lastname: Peters
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Becker
authors.forename: Jason D
authors.initials: JD
authors.lastname: Kahn
authors.forename: L James
authors.initials: LJ
authors.lastname: Maher
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D056968:Lac Repressors / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C116822:hns protein, E coli