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

Abstract

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.

DNA topology confers sequence specificity to nonspecific architectural proteins

“…Our recent studies of the structures of DNA loops mediated by the Lac repressor, the tetrameric protein assembly that controls the expression of lac genes in E. coli, show how the deformations of the double helix found in known high-resolution complexes of HU with DNA compensate for the intrinsic stiffness of short DNA (10,11). The random binding of the protein, at levels approximating those found in vivo, brings the predicted looping propensities in line with values deduced from geneexpression studies.…”

“…Although the spatial constraints placed on short DNA fragments looped between the binding sites of the Lac and Gal repressors differ from those associated with ring closure (10), the propensity for DNA to adopt a naturally straight double-helical form continues to determine the sites of preferential HU buildup on the spatially constrained duplex. The positioning of the architectural protein, in turn, influences the way in which DNA attaches to the loop-mediating protein (10,11). For example, in the absence of HU, DNA loops with the 92-bp wild-type spacing found in the lac operon tend to bind in antiparallel orientations to the binding headpieces of a V-shaped model of the Lac repressor (10,17,18).…”

A stochastic reaction-diffusion model for protein aggregation on DNA

“…[19][20][21][22][23][24][25][26][27] The fact that their coarse-grained model was based on elastic forces made the realization of a great number of atoms possible. 28 Their numerical simulations revealed a number of interesting physical mechanisms, including HU-assisted loop formation 24,26 and DNA-directed sequence speci¯city of nonspeci¯c bending proteins. 25 However, all the above mentioned models did not reveal the mechanism underlying the HU cooperative clustering and the related topological patterns.…”

Supercoiling biases the formation of loops involved in gene regulation

“…In addition, accessory proteins that modify the intervening DNA by adding kinks, and/or altering flexibility, shift the looping equilibrium. Theoretical studies estimated that kinks produced by heat-unstable (HU) protein binding enhanced looping of DNA segments 75-150 bp in length to levels comparable to those measured in vivo (Czapla et al 2013). In fact, HU-binding appears to generally enhance flexibility to the point that it masks the effect of intrinsic sequence-dependent flexibility on looping both in vitro and in vivo (Boedicker et al 2013;Becker et al 2007).…”