title: Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.
abstract: Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.
authors.affiliation: Discipline of Biochemistry, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: David G
authors.initials: DG
authors.lastname: Priest
authors.forename: Lun
authors.initials: L
authors.lastname: Cui
authors.forename: Sandip
authors.initials: S
authors.lastname: Kumar
authors.forename: David D
authors.initials: DD
authors.lastname: Dunlap
authors.forename: Ian B
authors.initials: IB
authors.lastname: Dodd
authors.forename: Keith E
authors.initials: KE
authors.lastname: Shearwin
mesh_terms: D000465:Algorithms; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007763:Lac Operon; D056968:Lac Repressors / Q000235:genetics*; D008962:Models, Theoretical; D011401:Promoter Regions, Genetic; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000235:genetics*; D013816:Thermodynamics; D013997:Time Factors; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins
keywords: TPM; j factor; promoter-enhancer