Specifically bound lambda repressor dimers promote adjacent non-specific binding

<div><p>Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.</p></div

Specifically bound lambda repressor dimers promote adjacent non-specific binding - Fig 3

<p><b>Variations in the numbers of particles (<i>a</i>) and counts per particle (<i>b</i>) as a function of CI protein concentration.</b> Squares (filled) and circles (open) correspond to WT DNA constructs containing the OL3 or OR3 sites, respectively.</p

Number of CI dimer that bind during the first (<i>n</i>_<i>1</i>) and second (<i>n</i>_<i>2</i>) transitions.

<p>Number of CI dimer that bind during the first (<i>n</i>_<i>1</i>) and second (<i>n</i>_<i>2</i>) transitions.</p

Variation in the amplitude of the mutant CI protein-DNA complex.

<p>The amplitudes of <i>a1</i> (filled squares) and <i>a2</i> (open circles) used to fit the auto-correlation data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194930#pone.0194930.e002" target="_blank">Eq 2</a>) are plotted as a function of mutant CI protein (D197G) concentration for (<i>a</i>) WT OL1, (<i>b</i>) WT OL3, or (<i>c</i>) WT OR3 DNA. For each DNA, <i>a</i>_<i>1</i> dropped from an initial maximum to a final minimum with a reciprocal rise in <i>a</i>_<i>2</i> as [CI] increased from 0 to 500 nM. In contrast to the wild-type protein, no third species developed at higher concentrations.</p

Specifically bound lambda repressor dimers promote adjacent non-specific binding - Fig 5

<p><b>Titrations of unlabeled DNA to assay competitive CI-binding to (<i>a</i>) wild-type OL3 or (<i>b</i>) wild-type OR3 DNA.</b> Increasing the concentration of unlabeled DNA (filled squares) from 0 to 0.3 μM in the solution used for FCS measurements reduced the fraction of CI bound. This decrease was characterized by a plateau for both OL3 and OR3. Clearly, competition by the unlabeled DNA progressively reduced the CI available for binding to the fluorescently labeled DNA. This is evidence against the presence of higher molecular weight complexes in which a head-to-head interaction of CI dimers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194930#pone.0194930.g001" target="_blank">Fig 1</a>) might stabilize FCS particles containing a labeled and an unlabeled DNA segment.</p

The CI concentrations at mid-points of the first and second transitions for titrations of CI with the indicated DNA constructs as shown in Figs 3 and 4.

<p>These are indicative measures of relative binding affinities for the different CI operators.</p

Regulation of bacteriophage gene expression by CI.

<p>(Upper) Tetramers, formed through lateral interactions of adjacent CI dimers (red circles) bound to high affinity “operator” (green) sites, inhibit transcription from the lytic promoters <i>pL</i> and <i>pR</i>. (Middle) The dimers bound at <i>OL</i>1 and <i>OL2</i> may interact head-to-head with those at <i>OR1</i> and <i>OR2</i>, to stabilize a DNA loop (dotted curve) to juxtapose and recruit dimers at the strong <i>OL3</i> and weaker OR3 sites which also interact head-to-head. The additional dimer on OR3 inhibits transcription from <i>pRM</i>. (Lower) The CI dimers at <i>OL3</i> and <i>OR3</i> are still available for lateral pairwise interaction and may cooperatively stabilize other dimers to adjacent, non-specific (gray) sites.</p

Measurement of positions and volumes for CI multimers which appeared as amorphous particles along DNA fragments containing OL and OR binding sites.

<p>(<i>a</i>) The positions of these protein particles were measured with respect to the nearest end at different concentrations. Even at the highest protein concentration almost all particles were found near 120 and 240 nanometers into the contour, where high affinity binding sites are located. (<i>b</i>) The sizes of CI particles increased with concentration as can be seen in a cumulative histogram of the volumes measured in scanning force nanographs recorded for DNA exposed to 200 (circles), 400 (crosses), or 800 (triangles) nM CI. (<i>c</i>) The D197G mutant CI protein also formed specifically positioned particles on the OL and OR bindings sites with little binding elsewhere. (<i>d</i>) However the particles of mutant protein, that does not form multimers larger than dimers, did not increase in size as a function of CI concentration.</p

Variation in the amplitude of the WT CI protein-DNA complex.

<p>The three amplitudes <i>a</i>_<i>1</i> (filled square), <i>a</i>_<i>2</i> (open circle), and <i>a</i>_<i>3</i> (open triangle) used to fit the auto-correlation data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194930#pone.0194930.e002" target="_blank">Eq 2</a>) are plotted as a function of CI protein concentration for (<i>a</i>) WT OL1, (<i>b</i>) WT OL3, or (<i>c</i>) WT OR3 DNA. Solid and dashed curves serve as guides for the transitions of <i>a</i>_<i>1</i> and <i>a</i>_<i>3</i> as the concentration of CI changes. For each DNA, <i>a</i>_<i>1</i> dropped from an initial maximum to a final minimum with a reciprocal rise in <i>a</i>_<i>2</i> as [CI] increased from 0 to 300 nM (<i>a</i>, <i>b</i>) or to 400 nM (<i>c</i>). As [CI] was increased further, <i>a</i>_<i>2</i> decreased to a minimum and <i>a</i>_<i>3</i> rose to maximum values at 500 nM CI.</p