Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information. © 2013 Czapla et al

DNA chain length controls the uptake of HU on LacR-mediated loops.

<p>The distribution of HU molecules bound to DNA loops mediated by the rigid, V-shaped LacR protein assembly varies regularly and abruptly with chain length <i>N</i>. The value of <i>f</i>_HU corresponds to the fraction of loops with the specified number of bound HU dimers. See the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone-0056548-g002" target="_blank">Figure 2</a>.</p

The composite interactions of DNA, LacR, and HU produce a multiplicity of looped states.

<p>Molecular snapshots reveal the complex interplay of protein and DNA structure in simulated LacR-mediated DNA with (A) 109 and (B) 115 base pairs between the centers of bound operators. Structures captured from computations performed in the absence and presence of randomly bound HU (upper and lower images in each set) and with allowance for opening of LacR from the V-shaped tetrameric assembly (right and left images in the sets). Images rendered with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) and drawn in a common viewing direction looking down the shortest principal axis of the C^α atoms in the LacR assembly, with the core of protein strand A always shown on the left. The DNA is represented by a color-coded backbone (5′ to 3′ chain progression depicted by the dark to light blue color change), and the protein by space-filled atomic representations (LacR in rose, HU in gold). The numbers below the images denote the fraction of loops with the given loop orientation and the illustrated number of bound HU proteins in the simulated ensembles. Addition (+) or removal (−) of HU and prohibition (Δ<i>α</i> = 0) or allowance (Δ<i>α</i> ≥0) for flexibility in LacR denoted along arrows.</p

DNA loops simulated in the presence of HU and deformable LacR capture <i>in-vivo</i> looping properties.

<p>The predicted ease of DNA loop formation between the headpieces of (A) a rigid V-shaped LacR complex and (B) a deformable LacR assembly mimics looping propensities deduced from the <i>in-vivo</i> expression of <i>lac</i> genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Becker1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Bond1" target="_blank">[13]</a>. The chain-length-dependent gene-expression profiles of <i>E. coli</i> cells are expressed as <i>J</i> factors using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548.e007" target="_blank">Eqn. (4)</a>. Values of <i>J</i> for a strain with wild-type (WT) proteins and a mutated strain (ΔHU) that cannot express HU are depicted respectively by large and small dark symbols, with data reported in different publications for the same DNA construct <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Becker1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Bond1" target="_blank">[13]</a> expressed as average values. The simulated <i>J</i> factors reflect the likelihood that an ideal, naturally straight DNA molecule folds along a pathway compatible with the spatial constraints imposed by the binding of LacR and the presence or absence of HU (points connected respectively by thick and thin lines).</p

DNA loop length and HU levels govern the opening of LacR.

<p>Contour surfaces of the likelihood of DNA loop formation as a function of chain length <i>N</i> and the change in the LacR opening angle Δ<i>α</i> reveal the deformations of the repressor induced by DNA chain length and suppressed by HU. Note the narrower range and lesser variation in Δ<i>α</i> found in loops (A) simulated in the presence of randomly bound HU compared to (B) those generated in the absence of the architectural protein. The average values of the opening angle at each chain length are reported in the line plots above the contour surfaces. The blue-to-red scale on the lower right denotes the probability of loop formation over the specified range of opening angles and chain lengths. Red denotes the more easily formed loops with higher <i>J</i> factors and blue the loops with lower <i>J</i> factors.</p

HU enhances the looping propensities of DNA.

<p>The probability of DNA looping between the headpieces of the rigid, V-shaped LacR assembly in the presence (+) or absence (−) of HU exceeds that of forming protein-free DNA minicircles of the same chain length <i>N</i>. The looping and cyclization propensities, or <i>J</i> factors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Jacobson1" target="_blank">[32]</a>, in (A) are obtained from the fraction of simulated configurations in ensembles of 10¹²–10¹⁶ fluctuating duplexes with chain ends in the requisite spatial disposition. The cartoon in (B), constructed using 3DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Lu1" target="_blank">[64]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Lu2" target="_blank">[65]</a> from the base-pair step parameters of successfully closed chains, illustrates the relative deformations of DNA entailed in cyclization compared to the formation of parallel and antiparallel loops of the same chain length (105 bp) and the different constraints on DNA ends. Configurations of HU-bound loops in A are generated such that there is one HU dimer randomly bound, on average, every 150 bp of DNA in the simulated ensembles. The double helix is assumed to be naturally straight in its equilibrium rest state, inextensible, and capable of isotropic bending and independent twisting at the base-pair level. The protein-bound DNA is modeled implicitly in terms of the base-pair-step parameters found in the currently best-resolved crystal structure of operator-bound LacR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Bell1" target="_blank">[31]</a> and the four high-resolution structures of DNA with <i>Anabaena</i> HU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Swinger1" target="_blank">[2]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Swinger3" target="_blank">[4]</a>. The shading of blocks in B denotes the minor-groove edges of the base pairs and the small arrows the end-to-end separation and direction of terminal base pairs. Note that the illustrated loops include only the (inner) halves of the bound operators counted in the chain length.</p

Molecular constraints imposed on protein-mediated DNA loops.

<p>(A) The non-specific architectural protein HU introduces a sharp bend in the DNA loop upon binding. (B) The Lac repressor assembly (LacR) sets the positions of the DNA operators at the ends of the loop. (C) The loop adopts one of four possible orientations on the LacR headpieces. The color-coding in B and C distinguishes the operator strands in terms of their contacts to the four LacR monomers: blue (strand A); red (strand B), yellow (strand C), green (strand D). The arrows denote the 5′-3′ directions of the operators on the binding headpieces, the characters A and P specify the antiparallel or parallel orientations of the bound operators, and the numerals 1 and 2 distinguish whether the first operator (O3) points toward the inside or outside of the assembly. The HU-bound DNA is represented by the best resolved crystal complex of the <i>Anabaena</i> protein with DNA (PDB entry 1P51) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Swinger1" target="_blank">[2]</a> and the LacR-DNA is a model obtained by composition <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Swigon1" target="_blank">[27]</a> of currently available X-ray data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Lewis1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Bell1" target="_blank">[31]</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#s4" target="_blank">Methods</a>). Note the very different bending of the protein-bound DNA, toward and around HU but away from the two LacR headpieces. Molecular images rendered with Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Pettersen1" target="_blank">[73]</a>.</p

LacR opening contributes to <i>in-vitro</i> DNA looping.

<p>Comparison of the simulated ease of LacR-mediated DNA loop formation in the absence of HU with the looping propensities deduced from tethered particle motion studies (filled-in circles bracketed by error bars) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone.0056548-Han1" target="_blank">[36]</a> hints of possible LacR opening <i>in vitro</i>. Simulated values are obtained from calculations with a rigid V-shaped protein complex (Δ<i>α</i> = 0) and a deformable LacR assembly(Δ<i>α</i> ≥0) and connected respectively by dashed and solid lines). See text for discussion of chain extension.</p

HU increases the mix of DNA loops formed on LacR.

<p>The types of DNA loops formed on the V-shaped LacR assembly underlie the chain-length-dependent variation in the <i>J</i> factor. Note the more complex plot of <i>J</i>(<i>N</i>) and the greater diversity of loops obtained in the presence (A) compared to the absence (B) of randomly bound HU molecules. The diversity is expressed in terms of the fraction of loops <i>f</i>_loop with DNA bound to LacR in one of the four specified orientations. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone-0056548-g001" target="_blank">Figure 1C</a> for schematics of the antiparallel (A1, A2) and parallel (P1, P2) forms and the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056548#pone-0056548-g002" target="_blank">Figure 2</a> for computational details. The wider range of <i>N</i> in (A) <i>vs</i>. (B) reflects the greater ease of forming short LacR-mediated loops in the presence of HU.</p