DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: Evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA

Kinetic studies of formation and dissociation of open-promoter complexes (RP,) involving E sigma(70) RNA polymerase (R) and the lambda P-R promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I-1 and I-2, 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 I-1 and I-2. At 0 degrees C, where extrapolation of equilibrium data indicates I-1 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 I-1. However, the DNA backbone in I, is extensively protected from DNase I cleavage; the DNase I footprint extends similar to 30 bases downstream and at least similar to 40 bases upstream from the start site. I, has a short lifetime (less than or equal to 15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degrees C to 0 degrees C, in the time-range where we conclude that the dominant, transiently accumulated complex:is I-2,DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I-1. However, unlike I-1, I-2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degrees C. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I-1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of E sigma(70). We propose that binding of the start site and downstream DNA in this clef 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 I-2. 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 degrees C, we obtain the equilibrium constant K-3 for the conversion of I-2 to RPo and the rate constant k(-2) for the conversion of I-2 to I-1 (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. (C) 1998 Academic Press.