Base-Specific Recognition of the Nontemplate Strand of Promoter DNA by E. coli RNA Polymerase  Jeffrey W Roberts, Christine W Roberts  Cell  Volume 86, Issue 3, Pages 495-501 (August 1996) DOI: 10.1016/S0092-8674(00)80122-1

Figure 1 Pathway of Open Complex Formation at σ70 Promoters (A) General scheme of open complex formation. The pathway can be written: R + P ⇄ RPc → RPi ⇄ RPo, where R and P designate RNA polymerase and promoter DNA, respectively (McClure 1985; Buc and McClure 1985). RPc is readily reversible to free R and P, allowing R to be inactivated by heparin. In contrast, conversion of RPo and RPi back to RPc and thence to free R is very slow, so that enzyme in RPi and RPc resists heparin challenge. (B) DNA sequences of the closed and open forms of the λ promoter pR′ and adjacent DNA (Kainz and Roberts 1992). RNA synthesis initiates at +1 and proceeds rightward, templated by the bottom (“template”) strand. Cell 1996 86, 495-501DOI: (10.1016/S0092-8674(00)80122-1)

Figure 2 Rate of Formation of the Heparin-Resistant Open Promoter Complex of pR′ DNAs Mutant in the −10 Region and Their Heteroduplexes with Wild-Type DNA Complexes were made during incubation at 25°C, sampled at intervals, and assayed by runoff synthesis at 37°C. (A) −12G. Runoff synthesis was quantitated by phosphorimager and is plotted in arbitrary units against time of incubation. (B) −11G: as for (A). (C) −7G: as for (A), except that the values were normalized (by less than 1.5-fold) to the same final value, in order to reveal more clearly the modest differences. Squares, homoduplex wild type; Xs, homoduplex mutant; triangles, nontemplate strand mutant heteroduplex; circles, template-strand mutant heteroduplex. (D) −11G: autoradiograph of the data plotted in (B). The paused RNAs (+16, +17) and the abortive RNAs (e.g., +12) of pR′ have been described (Yarnell and Roberts 1992; Ring and Roberts 1994; Ring et al. 1996). The time course of the pause is shown in Figure 4. Cell 1996 86, 495-501DOI: (10.1016/S0092-8674(00)80122-1)

Figure 3 Summary of Rates of Open Complex Formation for Seven Mutants and Their Heteroduplexes with Wild Type The data is taken from Table 1 presented as a rate (in arbitrary units) that is the reciprocal of the half-time for formation given in Table 1. Cell 1996 86, 495-501DOI: (10.1016/S0092-8674(00)80122-1)

Figure 4 Time Course and Nature of RNA Synthesized from Preformed Heparin-Resistant Complexes of the −11G Mutant Open complex was formed during incubation for 10 min at 37°C, heparin and MgCl2 were added, and synthesis reactions were sampled during incubation at 37°C. The relative moles RNA after the 8 min incubation, and hence the amount of open complex formed in 10 min at 37°C, was as follows: wild type, 1.0; −11G mutant, 0.19; nontemplate mutant heteroduplex, 0.84; template-strand mutant heteroduplex, 0.97. Note that the +16 pause has a much shorter half-life than the +17 pause, so that little is visible in Figure 2D. Quantification of this experiment also demonstrates that synthesis for 5 min, as was used for the experiments of Figure 2, Figure 3, and 5, is sufficient to accumulate all of the runoff. Cell 1996 86, 495-501DOI: (10.1016/S0092-8674(00)80122-1)

Figure 5 Dissociation of Preformed Open Complexes of −11G Mutant DNA, Wild-Type DNA, and the Two Heteroduplexes Complexes were formed by preincubation for 10 min at 37°C, heparin was added, and the temperature lowered to 17°C; samples were taken at intervals into MgCl2 and synthesis performed for 5 min at 37°C. Symbols are as in Figure 2. Cell 1996 86, 495-501DOI: (10.1016/S0092-8674(00)80122-1)