Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2007 Jun 15;93(8):2726–2731. doi: 10.1529/biophysj.107.111245

Probability of the Site Juxtaposition Determines the Rate of Protein-Mediated DNA Looping

Yury S Polikanov *, Vladimir A Bondarenko *, Vladimir Tchernaenko †, Yong I Jiang *, Leonard C Lutter †, Alexander Vologodskii ‡, Vasily M Studitsky *
PMCID: PMC1989718  PMID: 17573434

Abstract

Numerous biological processes are regulated by DNA elements that communicate with their targets over a distance via formation of protein-bridged DNA loops. One of the first questions arising in studies of DNA looping is whether the rate of loop formation is limited by diffusion of the DNA sites. We addressed this question by comparing the in vitro measured rates of transcription initiation in the NtrC-glnAp2 enhancer-dependent transcription initiation system with predictions of two different theoretical models. The promoter and enhancer were in a 7.6-kb plasmid and separated by 2.5 kb. The measurements were performed for different values of the plasmid superhelix density, from 0 to −0.07. Earlier theoretical analysis, based on the Monte Carlo simulation of DNA conformations, showed that if the rate of loop formation is determined by the equilibrium probability of juxtaposition of the DNA sites, the rate should be ∼100 times higher in supercoiled than in relaxed DNA. On the other hand, Brownian dynamics simulation showed that if the rate of loop formation is limited by the site diffusion, it should be nearly independent of DNA supercoiling. We found that efficiency of the transcription initiation increases by nearly two orders of magnitude as a result of the corresponding increase of the template supercoiling. This clearly shows that the rate of bridging in the enhancer-promoter system is not limited by diffusion of the DNA sites to one another. We argue that this conclusion derived for the specific system is likely to be valid for the great majority of biological processes involving protein-mediated DNA looping.

INTRODUCTION

Many processes of DNA metabolism involve interaction between or among two or more DNA sites widely separated along the molecule contour. Such processes require formation of loops by the DNA molecule (1). Loops are observed during DNA replication and repair (2), transcription regulation (3), genome rearrangements by site-specific recombinases (4), topological changes by topoisomerases (5), and action of many restriction enzymes (6). In at least some cases formation of the loops is a rate-limiting step for the entire process (7). Knowledge of the dynamic principles of DNA loop formation is important for better understanding of all these processes.

The process of loop formation can be divided into two steps: juxtaposition of two DNA sites and closing of the loop by a protein bridge. If the bridging is faster than separation of the juxtaposed sites by diffusion, the rate of loop formation is determined by the time of the first site juxtaposition, so the rate is limited by the rate of diffusion of the DNA sites to one another. Alternatively, if the separation of the juxtaposed sites by diffusion is faster than the protein bridging, many collisions of the sites precede stable bridging. In the latter case the rate of looping is proportional to the total fraction of time the sites are juxtaposed or to the equilibrium probability of the juxtaposition. Although many aspects of protein-mediated DNA looping have been studied both experimentally (see Halford et al. (6) for review) and theoretically (8–11), it has not been investigated which of two models better describes protein-mediated DNA looping.

The computer simulations showed that the equilibrium probability of juxtaposition of two selected sites in supercoiled DNA (scDNA) is ∼100 times higher than in the relaxed molecule, whereas the average time of the first juxtaposition of the same sites is nearly unaffected by supercoiling (9,10). These simulation results enable us to discriminate between the two mechanisms of bridging by studying the rate of bridging as a function of DNA supercoiling. In this work we studied the dynamics of loop formation during distant, enhancer-dependent activation of prokaryotic transcription.

Transcriptional enhancers are relatively short (30–200 bp) DNA sequences usually composed of several binding sites for activator protein(s) and are capable of activating transcription initiation from the promoters, which are often located at considerable distances in vivo (up to 60 kb in Eukaryota and up to 15 kb in Prokaryota; see Blackwood and Kadonaga (12) and Bondarenko et al. (13) for review). This activation is independent of enhancer orientation on DNA and of its position upstream or downstream of the promoter. Proteins bound at the enhancer and promoter directly interact with each other, forming a DNA loop (14,15). One of the most extensively studied prokaryotic enhancers is NtrC(NRI)-dependent, σ54-dependent transcriptional enhancer of E. coli that controls transcription from the glnAp2 promoter (see reviews (13,16,17)). This enhancer, consisting of two high-affinity NtrC-binding sites, can strongly activate transcription when positioned up to at least 3.5 kb in vitro (18,19). NtrC is an activator that binds to the enhancer and, after phosphorylation mediated by NtrB(NRII) protein, interacts with the Eσ54 RNA polymerase (RNAP) holoenzyme, bound at the promoter in form of the closed, inactive initiation complex (RPC) (Fig. 1). This enhancer-promoter (E-P) interaction activates conversion from the closed to the open (RPO) complex (20–23). Phosphorylation of NtrC also activates its ATPase activity, which is required to stimulate the RPC → RPO transition. During E-P interaction, the spacer DNA forms a loop (24,25).

FIGURE 1.

FIGURE 1

Open in a new tab

Diagram of the enhancer-dependent transcription activation of the glnAp2 promoter. (1) Phosphorylated NtrC activator is pre-bound at the enhancer (E) and is able to interact with closed complexes RPC preformed at the promoter (P). (2) Juxtaposition of the enhancer and promoter is accompanied by DNA looping and may result in (3) the E-P protein bridging; otherwise the two DNA-protein complexes may diffuse away from one another. The rate constant kbridging specifies the rate of bridging for DNA conformations with juxtaposed enhancer and promoter. Two models of the loop formation that are considered in the study correspond to the condition of kunloop ≫ kbridging and kunloop ≪ kbridging. The bridging involves a specific E-P protein-protein interaction and results in the formation of the transcription-competent RPO. It is important that under our experimental conditions this step is irreversible, so the number of protein-mediated E-P loops is equal to the number of RPO (7). (4) Adding heparin and labeled NTPs allows transcription from RPO. Because heparin prevents formation of new RPO (preformed RPO and the elongation complexes are resistant to heparin), the transcription is limited by one round. Thus, the number of transcripts is equal to the number of open complexes formed before addition of heparin (7).

In this work, the efficiencies of protein-mediated DNA looping in plasmids supercoiled to different superhelix densities were measured in vitro. Comparing these measurements with predicted dependences of the looping efficiency on DNA supercoiling for the two models, we concluded that the rate of loop formation between enhancer and promoter is determined by the probability of enhancer and promoter juxtaposition rather than the rate of their first collision.

MATERIALS AND METHODS

DNA and proteins

Negatively supercoiled pLY10 DNA template was purified as described (26). The promoter and enhancer of the 7.6-kb plasmid are separated by 2.5 kb.

All proteins and protein complexes were purified according to previously described protocols (27). Chicken blood extract (CBE) containing strong topoisomerase I activity was prepared as described previously (28).

Preparation of plasmid DNA with different superhelix densities

Negatively scDNA templates, purified from XL1-Blue strain of E. coli, were relaxed by incubation at 50 μg/ml with CBE (0.07 μl of CBE per 1 μg of plasmid DNA) in 200 mM NaCl, 20 mM Tris-HCl (pH 8.0), 0.25 mM EDTA, and 5% glycerol for 45 min at 37°C. To obtain plasmids that are negatively supercoiled to various extents, the pLY10 plasmid was relaxed in the presence of the following concentrations of chloroquine (μg/ml): 50 (σ = −0.01), 100 (σ = −0.022), 200 (σ = −0.035), 300 (σ = −0.046), 500 (σ = −0.056), 700 (σ = −0.069), and 1000 (σ = −0.074). The relaxed and partially supercoiled plasmids were then purified after proteinase K treatment (150 μg/ml) for 2 h at 37°C in the presence of 1% SDS, followed by phenol/chloroform extraction, ethanol precipitation, and passage through a Sephadex G-50 spin column. The levels of DNA supercoiling (σ) were determined after separation of the purified pLY10 topoisomers in agarose gels containing various concentrations of chloroquine (29). The obtained sets of pLY10 topoisomers have average superhelical densities σ of 0, −0.01, −0.022, −0.035, −0.046, −0.056, −0.069, and −0.074.

In vitro transcription

Conditions for in vitro transcription were optimized for maximal utilization of the pLY10 templates. Transcription was conducted as described (27) for 1 min to guarantee that fewer than 20% of templates were activated in the experiments, and the number of transcripts was proportional to the rate of formation of the E-P complexes.

Computer simulation

The Monte Carlo simulations were used to compute the probability of juxtaposition of DNA sites at different degrees of DNA supercoiling (9). In these simulations a DNA molecule is modeled as a chain consisting of rigid cylinders with elastic bending potential. The length of each cylinder corresponds to 10 nm, one-fifth of the chain persistence length. The cylinders are not interpenetrable, and their diameter, d, accounts both for the geometrical diameter of the double helix and for electrostatic repulsion between charged DNA segments. The value of d, 5 nm, corresponded to physiological ionic conditions. The torsional rigidity of DNA was equal to 3·10−19 erg·cm. The length of the model chain and separation between the analyzed sites corresponded to pLY10 plasmid. The time of the first collision of the two DNA sites was obtained by the Brownian dynamics (BD) simulations (10). Essentially the same discrete wormlike chain, with addition of the finite stretching rigidity, was used in the BD simulations. The hydrodynamic interaction between the model chain and solution and between the chain segments was also taken into account (10). All parameters of the model chain were identical for MC and BD simulations. Two chain segments were considered to be juxtaposed when the distance between their centers was <10 nm.

RESULTS

Intrinsic activity of the glnAp2 promoter does not depend on the level of DNA supercoiling

Supercoiling can change two features of DNA that, in turn, could affect the level of transcription. First, an increase of the level of negative supercoiling results in higher probability of basepair opening (30–32) that could modify the efficiency of transcription initiation from the glnAp2 promoter. This local, promoter-specific effect of DNA supercoiling could considerably change the rate of transcription (33,34). Second, DNA supercoiling changes tertiary conformations of DNA molecules, causing formation of branched interwound superhelices (see review (35)). In a highly supercoiled form of the pLY10 plasmid, the probability of juxtaposition of specific DNA sites can be ∼100 times higher than in the relaxed form (9), which could change the efficiency of E-P communication. Because in this study we wanted to address the second, global effect, the contribution of the local effect of DNA supercoiling on the transcription through the increased probability of basepair opening was evaluated first.

To learn whether the intrinsic activity of the glnAp2 promoter depends on the level of negative supercoiling, we explored the observation that in the absence of the enhancer glnAp2 promoter has a weak basal activity (18). Enhancer-independent transcription is specific and activator-dependent because it requires the presence of all other key components of enhancer-dependent transcription, including the activator NtrC, the protein kinase NtrB, σ54, and ATP (data not shown). The effect of negative DNA supercoiling on the intrinsic activity of the glnAp2 promoter was lower than 1.5-fold within the experimental range of σ-values (from 0 to −0.075, Fig. 2). Because negative DNA supercoiling can change the efficiency of enhancer-dependent, distantly activated transcription up to 50-fold (see below), we concluded that in our system supercoiling predominantly affects the transcription efficiency by changing global conformations of the plasmid DNA.

FIGURE 2.

FIGURE 2

Open in a new tab

Supercoiling does not significantly impact the intrinsic activity of the glnAp2 promoter. (A) Topoisomers of enhancerless pAN6 plasmid supercoiled to different extents were transcribed in the presence of an excess of NtrC protein. Transcripts were analyzed by denaturing polyacrylamide gel electrophoresis. (B) Intensities of the bands containing specific transcripts were quantified on a PhosphorImager and plotted as a function of the superhelical density of the plasmid DNA (the efficiency of transcription of native pAN6 (σ = −0.06) is 100%). All measurements were repeated twice; the standard deviation for the measured values of the promoter activity is close to 10%. The data show that the differences in supercoiling levels do not result in significant changes of the efficiency of enhancer-independent transcription from the glnAp2 promoter.

DNA supercoiling strongly facilitates the efficiency of enhancer-dependent promoter activation

Next, the rates of protein-mediated DNA looping were measured using an enhancer-dependent transcription assay (Fig. 1). We prepared a set of samples of pLY10 plasmid with different values of negative superhelix density, σ (see Methods). Then the efficiencies of transcription of differently supercoiled pLY10 templates were compared in a single-round in vitro transcriptional assay (see Liu et al. (7) for details). The templates were preincubated with all components of the transcription machinery. This results in binding of NtrC activator to the enhancer and formation of the closed, inactive RPC on the glnAp2 promoter (18,36,37). The NtrC activator cannot interact with the RPC and activate transcription unless it is phosphorylated by the NtrB protein, which is also present in the reaction but was inactive in the absence of ATP (24,25). Then ATP was added for a limited time (1 min) to provide the substrate for phosphorylation of the NtrC activator. NtrC phosphorylation makes possible E-P protein-mediated interaction and formation of the transcriptionally active RPO. Subsequent addition of nucleotides together with heparin allows completion of initiated transcripts and prevents formation of new initiation complexes, resulting in a single-round transcription from each activated promoter. The majority of RNA polymerase molecules terminate after formation of 401-nt RNA transcript (7). Thus, the readout of the assay (the amount of 401-nt transcripts) reflects the number of the open initiation complexes formed, which is equivalent to the rate of the protein-mediated E-P looping (7). Of course, the equivalence is valid only under the condition that a small fraction of glnAp2 promoters is activated during the experiments. Therefore, the experimental conditions were selected with a reaction condition to allow formation of open complexes on <20% of the templates.

The results of the experiments show that efficiency of enhancer-dependent, distantly activated transcription greatly increases as the level of negative supercoiling of the pLY10 plasmid changes from 0 to −0.075 (Fig. 3). Over the entire interval of σ this increase constitutes nearly two orders of magnitude. Thus, the rate of the protein-mediated E-P looping (bridging) is dramatically increased by negative DNA supercoiling of the plasmid.

FIGURE 3.

FIGURE 3

Open in a new tab

Effect of DNA supercoiling on the rate of enhancer-dependent transcription activation and its comparison with theoretical models. (A) The labeled transcripts, synthesized on supercoiled plasmid pLY10 with different values of σ. The transcripts were separated by denaturing polyacrylamide gel electrophoresis. (B) Intensities of the bands containing the specific 401-nt transcript were quantified on a PhosphorImager and are shown by open circles. The standard deviations for the measured values of the promoter activity are close to 20%. The solid line shows the dependence of the juxtaposition probability on σ; the rate of the first juxtaposition as a function of σ is shown by the dashed line. Both theoretical dependences are shown in arbitrarily chosen relative units, so only the changes of the corresponding values with σ should be compared with the experimental data. The standard deviations for both theoretical dependencies are close to 10%. The theoretical dependences were obtained for the plasmid pLY10 used in the experiments.

Probability of E-P juxtaposition determines the efficiency of transcription activation

We compared the experimental results on the efficiency of transcription activation with the theoretical predictions for two models of the protein-mediated DNA looping. The first model assumes that the rate of loop formation is not limited by diffusion of the promoter and enhancer into close proximity to each other. In this case many collisions of the promoter and enhancer precede formation of the loop, and the rate of looping is proportional to the equilibrium probability of the site juxtaposition. This probability strongly increases as a function of |σ| (9). We recalculated the probability as function of σ for contour length and E-P separation in the pLY10 plasmid (Fig. 3 B, solid line). The second model assumes that the rate of loop formation is limited by the rate of diffusion of one site to another, so the first juxtaposition of the sites results in the loop formation. Brownian dynamics simulation shows that the average time of the first juxtaposition is nearly independent of |σ| (10). This simulation was used to obtain the corresponding dependence for pLY10 plasmid (Fig. 3 B, dashed line). Clearly, the experimental data are consistent only with the first model suggesting that the rate of loop formation is not limited by the rate of the site diffusion.

DISCUSSION

Supercoiled DNA molecules form interwound branched superhelices. In such a conformation, many pairs of sites, separated along the DNA contour, are positioned in close vicinity to each other. In other words, in each conformation of scDNA there are many pairs of juxtaposed sites. Typical conformation of relaxed DNA of the same size is characterized by a much smaller number of juxtaposed sites. Therefore, the probability of juxtaposition for any pair of sites is ∼2 orders of magnitude higher in scDNA than in relaxed DNA (9). The corresponding data, obtained by the Monte Carlo simulations, perfectly matches the measured dependence of the transcription efficiency on DNA supercoiling (Fig. 3 B). On the other hand, it is known from the Brownian dynamics computer simulation that the average time of the first juxtaposition of two sites is hardly affected by DNA supercoiling (10). The latter result derives from the fact that supercoiling slows down the exchange between different DNA conformations (10). Comparison between the experimental and theoretical data clearly shows that formation of a DNA loop, mediated by the E-P interaction, is not a diffusion-limited process. The first collision of the specific sites does not result, typically, in protein bridging. The rate of bridging is proportional to the fraction of time the sites are juxtaposed or to the probability of their juxtaposition.

We studied here only one specific system of protein-mediated DNA looping. We think, however, that our conclusion should be valid for the great majority of such systems. Indeed, the conclusion means that reorientations and repeating collisions of the juxtaposed DNA sites, often bound with proteins, are not fast enough to provide the bridging during a single juxtaposition event before the sites diffuse away one from another. These relative rates of site reorientation and separation cannot be changed dramatically because both of them are essentially specified by the internal flexibility of DNA molecules (this is not true for the juxtaposition probability, which is affected by many factors). The double helix is a very rigid molecule, and, therefore, any movement of the sites involves relatively large DNA segments. Thus, changing the size of bound proteins should not change essentially the size of the complex, which moves as a rigid body. Correspondingly, it should not change the rates of separation and reorientation of the juxtaposed DNA sites. For the same reason, the rates of separation and reorientation of the juxtaposed sites should not be affected by the spacing between them. The bridging of the juxtaposed sites could occur faster if it is mediated by very flexible proteins, attached to the DNA sites. In this case the protein bridging can occur for a wider range of the mutual orientations and distances between the sites because of faster motion of the protein interfaces. However, there are data that suggest that even increased flexibility of the bridging proteins can hardly make DNA looping a diffusion-limited process. It has been shown that protein-free DNA cyclization, which occurs by joining long cohesive ends, is not a diffusion-limited process (38), although the single-stranded cohesive ends are very flexible. Therefore, we believe that the conclusion, which was drawn for a particular E-P system, is valid in the general case of protein-mediated DNA looping in vitro. Our analysis, combined with other available data, strongly suggests that protein-mediated DNA looping is not a diffusion-limited process. Of course, inside the cell DNA is bound with chromatin proteins, which completely change DNA conformational properties, and our analysis cannot be extended to this case.

Acknowledgments

The work was supported by National Science Foundation grant 0549593 to V.M.S. and National Institutes of Health grant GM54215 to A.V.

Yong I. Jiang's present address is Dept. of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.

Editor: David P. Millar.

References

  • 1.Wang, J. C., and G. N. Giaever. 1988. Action at a distance along a DNA. Science. 240:300–304. [DOI] [PubMed] [Google Scholar]
  • 2.Benkovic, S. J., A. M. Valentine, and F. Salinas. 2001. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70:181–208. [DOI] [PubMed] [Google Scholar]
  • 3.Ogata, K., K. Sato, and T. H. Tahirov. 2003. Eukaryotic transcriptional regulatory complexes: cooperativity from near and afar. Curr. Opin. Struct. Biol. 13:40–48. [DOI] [PubMed] [Google Scholar]
  • 4.Nash, H. A. 1996. Site-specific recombination: integration, excition, resolution and inversion of defined DNA segments. In Escherichia coli and Salmonella. F. C. Neidhardt, editor. American Society for Microbiology, Washington DC. 2330–2376.
  • 5.Wang, J. C. 1996. DNA topoisomerases. Annu. Rev. Biochem. 65:635–695. [DOI] [PubMed] [Google Scholar]
  • 6.Halford, S. E., A. J. Welsh, and M. D. Szczelkun. 2004. Enzyme-mediated DNA looping. Annu. Rev. Biophys. Biomol. Struct. 33:1–24. [DOI] [PubMed] [Google Scholar]
  • 7.Liu, Y., V. Bondarenko, A. Ninfa, and V. M. Studitsky. 2001. DNA supercoiling allows enhancer action over a large distance. Proc. Natl. Acad. Sci. USA. 98:14883–14888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Allemand, J. F., S. Cocco, N. Douarche, and G. Lia. 2006. Loops in DNA: An overview of experimental and theoretical approaches. Eur. Phys. J. 19:293–302. [DOI] [PubMed] [Google Scholar]
  • 9.Vologodskii, A., and N. R. Cozzarelli. 1996. Effect of supercoiling on the juxtaposition and relative orientation of DNA sites. Biophys. J. 70:2548–2556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Huang, J., T. Schlick, and A. Vologodskii. 2001. Dynamics of site juxtaposition in supercoiled DNA. Proc. Natl. Acad. Sci. USA. 98:968–973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Klenin, K. V., and J. Langowski. 2001. Diffusion-controlled intrachain reactions of supercoiled DNA: Brownian dynamics simulations. Biophys. J. 80:69–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Blackwood, E. M., and J. T. Kadonaga. 1998. Going the distance: a current view of enhancer action. Science. 281:61–63. [DOI] [PubMed] [Google Scholar]
  • 13.Bondarenko, V. A., Y. V. Liu, Y. I. Jiang, and V. M. Studitsky. 2003. Communication over a large distance: enhancers and insulators. Biochem. Cell Biol. 81:241–251. [DOI] [PubMed] [Google Scholar]
  • 14.Rippe, K., P. H. von Hippel, and J. Langowski. 1995. Action at a distance: DNA-looping and initiation of transcription. Trends Biochem. Sci. 20:500–506. [DOI] [PubMed] [Google Scholar]
  • 15.Gaudreau, L., A. Schmid, D. Blaschke, M. Ptashne, and W. Horz. 1997. RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter. Cell. 89:55–62. [DOI] [PubMed] [Google Scholar]
  • 16.Rombel, I., A. North, I. Hwang, C. Wyman, and S. Kustu. 1998. The bacterial enhancer-binding protein NtrC as a molecular machine. Cold Spring Harb. Symp. Quant. Biol. 63:157–166. [DOI] [PubMed] [Google Scholar]
  • 17.Buck, M., M. T. Gallegos, D. J. Studholme, Y. Guo, and J. D. Gralla. 2000. The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor. J. Bacteriol. 182:4129–4136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Popham, D. L., D. Szeto, J. Keener, and S. Kustu. 1989. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 243:629–635. [DOI] [PubMed] [Google Scholar]
  • 19.Bondarenko, V. A., Y. I. Jiang, and V. M. Studitsky. 2003. Rationally designed insulator-like elements can block enhancer action in vitro. EMBO J. 22:4728–4737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Porter, S. C., A. K. North, A. B. Wedel, and S. Kustu. 1993. Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. Genes Dev. 7:2258–2273. [DOI] [PubMed] [Google Scholar]
  • 21.Wedel, A., and S. Kustu. 1995. The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation. Genes Dev. 9:2042–2052. [DOI] [PubMed] [Google Scholar]
  • 22.Wyman, C., I. Rombel, A. K. North, C. Bustamante, and S. Kustu. 1997. Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science. 275:1658–1661. [DOI] [PubMed] [Google Scholar]
  • 23.Hwang, I., T. Thorgeirsson, J. Lee, S. Kustu, and Y. K. Shin. 1999. Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. Proc. Natl. Acad. Sci. USA. 96:4880–4885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Su, W., S. Porter, S. Kustu, and H. Echols. 1990. DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc. Natl. Acad. Sci. USA. 87:5504–5508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Rippe, K., M. Guthold, P. H. von Hippel, and C. Bustamante. 1997. Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase × sigma 54 holoenzyme by scanning force microscopy. J. Mol. Biol. 270:125–138. [DOI] [PubMed] [Google Scholar]
  • 26.Rubtsov, M. A., Y. S. Polikanov, V. A. Bondarenko, Y. H. Wang, and V. M. Studitsky. 2006. Chromatin structure can strongly facilitate enhancer action over a distance. Proc. Natl. Acad. Sci. USA. 103:17690–17695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bondarenko, V., Y. V. Liu, A. J. Ninfa, and V. M. Studitsky. 2003. Assay of prokaryotic enhancer activity over a distance in vitro. Meth. Enzymol. 370:324–337. [DOI] [PubMed] [Google Scholar]
  • 28.Camerini-Otero, R. D., and G. Felsenfeld. 1977. Supercoiling energy and nucleosome formation: the role of the arginine-rich histone kernel. Nucleic Acids Res. 4:1159–1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Tchernaenko, V., H. R. Halvorson, L. C. Lutter, M. Radlinska, C. Drabik, and J. Bujnicki. 2003. Topological measurement of an A-tract bend angle: variation of duplex winding. Topological measurement of an A-tract bend angle: comparison of the bent and straightened states. J. Mol. Biol. 326:751–760. [DOI] [PubMed] [Google Scholar]
  • 30.Dean, W. W., and J. Lebowitz. 1971. Partial alteration of secondary structure in native superhelical DNA. Nat. New Biol. 231:5–8. [PubMed] [Google Scholar]
  • 31.Vologodskii, A. V., A. V. Lukashin, V. V. Anshelevich, and M. D. Frank-Kamenetskii. 1979. Fluctuations in superhelical DNA. Nucleic Acids Res. 6:967–982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Bauer, W. R., and C. J. Benham. 1993. The free energy, enthalpy and entropy of native and of partially denatured closed circular DNA. J. Mol. Biol. 234:1184–1196. [DOI] [PubMed] [Google Scholar]
  • 33.Lim, H. M., D. E. Lewis, H. J. Lee, M. Liu, and S. Adhya. 2003. Effect of varying the supercoiling of DNA on transcription and its regulation. Biochemistry. 42:10718–10725. [DOI] [PubMed] [Google Scholar]
  • 34.Peter, B. J., J. Arsuaga, A. M. Breier, A. B. Khodursky, P. O. Brown, and N. R. Cozzarelli. 2004. Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli. Genome Biol. 5:R87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Vologodskii, A. V., and N. R. Cozzarelli. 1994. Conformational and thermodynamic properties of supercoiled DNA. Annu. Rev. Biophys. Biomol. Struct. 23:609–643. [DOI] [PubMed] [Google Scholar]
  • 36.Buck, M., and W. Cannon. 1992. Activator-independent formation of a closed complex between sigma 54- holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae. Mol. Microbiol. 6:1625–1630. [DOI] [PubMed] [Google Scholar]
  • 37.Bondarenko, V., Y. Liu, A. Ninfa, and V. M. Studitsky. 2002. Action of prokaryotic enhancer over a distance does not require continued presence of promoter-bound sigma54 subunit. Nucleic Acids Res. 30:636–642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wang, J. C., and N. Davidson. 1968. Cyclization of phage DNAs. Cold Spring Harb. Symp. Quant. Biol. 33:409–415. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES