SINGLE MOLECULE FRET OF LACI-DNA-IPTG LOOP CONFORMATIONS

Abstract

This work focuses on the <italic>Escherichia coli</italic> lactose repressor protein (LacI) which represses expression of the lac operon. In order to repress transcription, the tetrameric LacI protein binds a primary promoter-proximal operator, O₁, and one of two auxiliary operators, O₂ or O₃. The binding of these two sites to a single LacI molecule occurs via DNA loop formation. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. The synthetic inducer isopropyl-&beta;-D-thiogalactoside (IPTG) acts similarly to allolactose. Model DNA constructs have been used to demonstrate, through fluorescence resonance energy transfer (FRET) analysis, that LacI may change conformation in order to form more than one loop structure. This work employs single molecule FRET to investigate LacI-induced loop formation in DNA looping constructs, as a function of IPTG concentration, on freely diffusing LacI-DNA complexes. The results include evidence for the persistence of DNA loop formation under at saturating IPTG concentration, and they provide a detailed view of how LacI conformation affects DNA loop formation. In addition, this work explores possible changes in geometry in LacI-induced DNA loops through the use of model DNA constructs that produce alternative loop topologies. We propose that inducer-bound LacI-DNA looped complexes may control the kinetics of induction and re-repression of the operon.