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. 2001 Dec 11;98(26):14883–14888. doi: 10.1073/pnas.261477898

Figure 3.

Figure 3

DNA supercoiling strongly facilitates enhancer–promoter communication over a large distance. (A) Time courses of enhancer–promoter communication on relaxed and sc plasmids with 0.11- or 2.5-kb enhancer–promoter spacing. Analysis of labeled transcripts in denaturing PAGE. The experimental strategy for comparison of the rates of enhancer–promoter communication on relaxed (Topo I +) and sc (Topo I −) plasmids with 0.11- or 2.5-kb enhancer–promoter spacing is outlined at the top. Sc plasmids were incubated in the presence or in the absence of topo I to obtain relaxed or supercoiled products, respectively. Then the templates were incubated in the presence of all components of the reaction except ATP to allow binding of the polymerase and NtrC. ATP was added to the reaction to allow functional enhancer–promoter communication, and the rate of promoter opening was measured by using a single-round transcription assay. The control pAN6 plasmid does not contain the enhancer. (B) The rate of enhancer–promoter communication is much lower when enhancer–promoter spacing is large (2.5 kb) and DNA is relaxed. Quantitative analysis of the data shown in A. The intensities of the bands containing 484-nt transcripts were analyzed by using a PhosphorImager. Transcription is saturated after three or four rounds, probably because ATP pool is depleted by NtrC.