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Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Eσ70 RNA polymerase (R) and the λPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0°C, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends ∼30 bases downstream and at least ∼40 bases upstream from the start site. I1 has a short lifetime (⩽15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37°C to 0°C, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0°C. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Eσ70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0°C, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k−2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures. |
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