DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

No abstract

The E. coli Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Single molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. FRET histograms suggest that the remaining population is a mixture of lower-FRET states ascribed to specificnonspecific or extended LacI loops, not free DNA. IntroductionThe control of the lac operon in E. coli by Lac repressor (LacI) is a classic example of negative gene regulation. The LacI homotetramer (a dimer of dimers) has the ability to bind two operator sites simultaneously, forming a DNA loop. In part, the biological function of looping is that binding of one dimer to its operator increases the effective local concentration of the other operator around the other dimer, leading to increased occupancy of the second operator.Probing the role of inducers, anti-inducers, and other allosteric effectors is important in understanding the regulation of protein-DNA looped complexes. In the presence of the artificial inducer, isopropyl-β, Dthiogalactoside (IPTG), there is about a 1000-fold decrease in the affinity of LacI for the operator. One IPTG molecule binds to the core domain of one monomer of LacI. The reorientation of the core domain increases the distance between DNA binding headpieces in a dimer, destabilizing the strong interactions between the headpiece and the DNA operator (figure 1).Our interest is in mutual interactions among inducer binding, DNA site selection, and DNA loop stability, and therefore we focus here on conditions where inducer is saturating, the DNA is designed to form exceptionally stable loops, and the protein:DNA ratio is ~1. A complete quantitative understanding of the lac operon, and by extension all genes regulated by ligand-responsive DNA looping proteins, will require confronting a more complete structural and thermodynamic characterization of all of the possible protein-DNAeffector complexes. SM-FRET on freely diffusing LacI-DNA loops allows us to analyze population distributions directly as opposed to the average properties measured in a bulk experiment, and the efficiency of energy transfer should be highly sensitive to changes in loop geometry.

No abstract