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DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

ABSTRACT: Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.

SUBMITTER: Hirsh AD

PROVIDER: S-EPMC3145271 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

Hirsh Andrew D AD Lillian Todd D TD Lionberger Troy A TA Perkins N C NC

Biophysical journal 20110801 3

Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformatio ...[more]

PMID: 21806940

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Project description:Thermodynamic analysis of urea-biopolymer interactions and effects of urea on folding of proteins and alpha-helical peptides shows that urea interacts primarily with polar amide surface. Urea is therefore predicted to be a quantitative probe of coupled folding, remodeling, and other large-scale changes in the amount of water-accessible polar amide surface in protein processes. A parallel analysis indicates that glycine betaine [N,N,N-trimethylglycine (GB)] can be used to detect burial or exposure of anionic (carboxylate, phosphate) biopolymer surface. To test these predictions, we have investigated the effects of these solutes (0-3 m) on the formation of 1:1 complexes between lac repressor (LacI) and its symmetric operator site (SymL) at a constant KCl molality. Urea reduces the binding constant K(TO) [initial slope dlnK(TO)/dm(urea) = -1.7 +/- 0.2], and GB increases K(TO) [initial slope dlnK(TO)/dm(GB) = 2.1 +/- 0.2]. For both solutes, this derivative decreases with an increase in solute concentration. Analysis of these initial slopes predicts that (1.5 +/- 0.3) x 10(3) A2 of polar amide surface and (4.5 +/- 1.0) x 10(2) A2 of anionic surface are buried in the association process. Analysis of published structural data, together with modeling of unfolded regions of free LacI as extended chains, indicates that 1.5 x 10(3) A2 of polar amide surface and 6.3 x 10(2) A2 of anionic surface are buried in complexation. Quantitative agreement between structural and thermodynamic results is obtained for amide surface (urea); for anionic surface (GB), the experimental value is approximately 70% of the structural value. For LacI-SymL binding, two-thirds of the structurally predicted change in amide surface (1.0 x 10(3) A2) occurs outside the protein-DNA interface in protein-protein interfaces formed by folding of the hinge helices and interactions of the DNA binding domain (DBD) with the core of the repressor. Since urea interacts principally with amide surface, it is particularly well-suited to detect and quantify the extent of coupled folding and other large-scale remodeling events in the steps of protein-nucleic acid interactions and other protein associations.

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DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

Publications

DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

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