Interactions of RNA polymerase with promoters can vary with temperature (Cowing et al. 1989; Schickor et al. 1990; Mecsas et al. 1991), for example, the transition from closed to open complexes requires temperatures above 20°C. Therefore, we investigated the interactions of RNAP with Pm in the absence of Mor over a range of temperatures. Figure 4A shows that Pm also follows this general rule. In the presence of both Mor and RNAP open complex formation, assayed by permanganate footprinting, occurred well at 30°C (Fig. 4A, lane 13), not at all at 5°C (Fig. 4A, lane 4), and extremely poorly, if at all, at 15°C (Fig. 4A, lane 8). To examine the temperature dependence of the −51 hypersensitivity, we carried out DNase I footprinting at 5°C, 15°C, and 30°C so we could compare the band patterns at low temperature to those of open complexes produced at 30°C. Binding of Mor alone, as assayed by Mor footprint formation, occurred equally well at all three temperatures (Fig. 4B, lanes 2, 7, and 12). Binding of RNAP in the absence of Mor, as assayed by position −51 hypersensitivity, also occurred well at all three temperatures (Fig. 4B, dots identify position −51 in lanes 3, 8, and 13). As expected, incubation of Mor and RNAP with Pm DNA at 5°C and 15°C did not produce the RNAP-dependent downstream footprints from −34 to +14 characteristic of open complexes; whereas heparin-resistant open complexes were formed in reactions incubated at 30°C (Fig. 4B, lanes 14 and 15). Note that the RNAP αCTD footprints from −59 to −61 upstream of bound Mor were generated equally well at all three temperatures (Fig. 4B, lanes 4, 9, and 14), indicating that they arose independently of open complex formation, and most likely reflected the presence of closed complexes formed at 5°C and 15°C. The upstream footprints observed at 5°C and 15°C were abolished by addition of heparin (Fig. 4B, lanes 5 and 10), supporting the hypothesis that they reflected the presence of closed complexes. In contrast, the Mor footprint, and therefore Mor binding, was unaffected by the addition of heparin (Fig. 4B, lanes 5 and 10). The presence of the upstream footprint in the absence of heparin at 5°C and 15°C demonstrated that RNAP could bind to Pm in the presence of Mor, even in the absence of open complex formation (Fig. 4B, lanes 4 and 9). With RNAP alone, the −51 hypersensitivity occurred at all three temperatures, and thus, is Mor-independent and does not require open complex formation. We propose that it arises by a transient interaction of RNAP with Pm. In contrast, when both RNAP and Mor were present, the −51 hypersensitivity was prevented by Mor binding; instead a footprint just upstream of Mor arose at all three temperatures, which we conclude is due to the presence of heparin-sensitive closed complexes at 5°C and 15°C and heparin-resistant open complexes at 30°C. The clarity of the upstream footprint, presumably caused by αCTD binding, demonstrated that RNAP binding in the presence of Mor was quite strong, in essence we propose, using Mor-RNAP interactions to tether RNAP to the promoter in the absence of stable RNAP −10 interaction.
Figure 4. DNase I and KMnO4 footprinting at different temperatures. Binding reactions were generated with probe containing Pm sequence −98 to +46 and flanking vector DNA, as described for Figure 3 except that samples were incubated at 5°C, 15°C, and 30°C. For KMnO4 footprinting in panel (A) the probe was labeled at the 5′ end of the bottom strand. After 5 min incubation with Mor and 6 min incubation with RNAP, the samples were subjected to KMnO4 modification and cleavage. Arrowheads mark promoter positions of specific G-ladder bands. For DNase I footprinting in panel (B) lanes 5, 10, and 15 also received heparin to 100 ng/μL, and the mixture was incubated for 1 min prior to DNase I digestion. Bars indicate the extent of the footprints generated by Mor alone, or by Mor and RNAP together. Arrowheads indicate the promoter positions of G-ladder bands. Dots mark the bands at position −51. Arrows and arrowheads marked HS identify positions −25, −57, −58 that are hypersensitive to DNase I digestion.
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