Insights into genome architecture deduced from the properties of short Lac repressor-mediated DNA loops

Abstract

Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allows for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein-DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains.

Synergy between Protein Positioning and DNA Elasticity: Energy Minimization of Protein-Decorated DNA Minicircles

“…The results for both proteins are presented in Figure . The relaxed minicircles include configurations from two competing families of structures, which exhibit similar ∼21 bp periodic dependencies on chain length. − The oscillations of energy with chain length differ in phase by about a helical turn such that the valleys in the energy profile of one family of closed configurations coincide with the peaks in the other and vice versa. The breaks in the plotted data correspond to jumps between the configurational families.…”

Designed architectural proteins that tune DNA looping in bacteria

“…In the absence of knowledge of the directions in which the DNA operators attach to the arms of the repressor, each operator is placed in two orientations on the protein-binding headpieces, yielding four distinct loops — two termed A 1 and A 2 with the 5′-3′ directions of the bound fragments running in nearly opposing (antiparallel) directions and two termed P 1 and P 2 with the fragments running in the same (parallel) direction (52). Moreover, each of these loops includes configurations from two competing structural families, with similar, albeit out-of-phase, dependencies on chain length (53,54), leading to eight potential spatial forms , etc. distinguished by the family (F1, F2) and connectivity (1, 2) of the loop.…”

“…Optimized structures of DNA loops of increasing chain length are obtained from the configurations of previously determined protein-free loops with 92-bp center-to-center operator spacing (54). Base pairs are added one at a time by assigning the coordinates of an arbitrary base pair in an existing structure to a new residue and then minimizing the energy of the enlarged system under the same end-to-end constraints.…”

Designed architectural proteins that tune DNA looping in bacteria

“…In the absence of knowledge of the directions in which the DNA operators attach to the arms of the repressor, each operator is placed in two orientations on the protein-binding headpieces, yielding four distinct loops (Figure 2 DE)—two termed A 1 and A 2 with the 5′-3′ directions of the bound fragments running in nearly opposing (antiparallel) directions and two termed P 1 and P 2 with the fragments running in the same (parallel) direction ( 62 ). Moreover, each of these loops includes configurations from two competing topological families, with similar, albeit out-of-phase, dependencies on chain length ( 63 , 64 ), leading to eight potential spatial forms \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\rm{A}}_1^{{\rm{F1}}}{\rm{,A}}_1^{{\rm{F2}}},{\rm{A}}_2^{{\rm{F1}}}$\end{document} , etc. distinguished by the family (F1, F2) and connectivity (1,2) of the loop.…”

“…Optimized structures of DNA loops of increasing chain length are obtained from the configurations of previously determined protein-free loops with 92-bp center-to-center operator spacing ( 64 ). Base pairs are added one at a time by assigning the coordinates of an arbitrary base pair in an existing structure to a new residue and then minimizing the energy of the enlarged system under the same end-to-end constraints.…”