Flexibility and Control of Protein–dna Loops

Abstract

Protein-DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)-DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI-DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI-DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than would be expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI-DNA structure is strongly influenced by flanking DNA.

Computational Analysis of Looping of a Large Family of Highly Bent DNA by LacI

“…By contrast, electron microscopy and x-ray scattering demonstrate a flexible V-region in solution (29,30). DNA looping models that include approximate treatments of protein flexibility align reasonably well with experimental data (16,26,31) and thereby support a flexible V.…”

“…The experimental and computational data for the three synthesized sequences already provide evidence for two distinct looped states: qualitatively different topoisomer distributions and FRET behavior have been observed for the 9C14 and 11C12 molecules. The existence of the two states was proposed to arise primarily from flexibility of LacI at the base of the ''V'' (3,26,39). The results presented here and in Goyal et al (12) suggest an alternative explanation, that distinct FIGURE 6 Computed minicircles mimicking the experimental procedure of Mehta and Kahn (3).…”

“…A multiscale method which couples a rod formulation for the DNA with an MD formulation of LacI has been presented (23,24). Potentially limiting the rod model is its exclusion of entropic effects, which are otherwise included in statistical approaches (see, for example, (21,25,26)). However, an extension of the Shimada-Yamakawa theory for DNA cyclization (27) leads to a computational method for evaluating the entropic contribution to the free energy of looping (28).…”

Gene repression by minimal lac loops in vivo

“…Figure 6 B illustrates LacI binding operators in the apical loop of superhelical DNA. A putative ‘open form’ of the repressor is shown, for which there is independent evidence ( 27 , 62 , 63 ). The blue protein illustrates one dimer of LacI rotated away from the other, anchored by the four-helix bundle tetramerization domain [as suggested by Steitz and coworkers ( 64 )].…”

Supercoiling Effects on Short-Range DNA Looping in E. coli

“…The E . coli lac operon provides a classic model for understanding control of gene expression by DNA looping [5–10] and for measuring DNA flexibility in vivo [11–14]. Lac repressor protein (LacI) is a tetramer that controls gene expression through its ability to simultaneously bind pairs of operator sites on DNA.…”