Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA : Measurements of the rate of open complex formation at T7 promoter A 1

Abstract

We have studied the rate of open promoter complex formation in vitro between Escherichia coli RNA polymerase holoenzyme and the A 1 promoter on bacteriophage T7 DNA, employing a novel method based on the quantitative transcription assay of Chamberlin et al. (1979). The procedure measures the time needed to achieve a constant rate of RNA synthesis in a reaction in which each RNA polymerase molecule is restricted to a single round of transcription. This time is inversely proportional to the rate of open complex formation by active RNA polymerase molecules, provided that RNA chain initiation is sufficiently rapid. Under solution conditions that favor formation of open promoter complexes at T7 promoter A 1 at equilibrium, the rates observed are in good agreement with those obtained using other methods such as filter binding, rifampicin challenge or template competition. However, the procedure is also applicable in many situations where other methods cannot be used. This includes reaction conditions such as low temperatures or elevated ionic strengths, where open promoter complexes are not stable. Studies using this procedure, taken with measurements based on template competition experiments (Kadesch et al., 1982, accompanying paper) yield a general picture of the major steps in the binding and activation of RNA polymerase at the T7 A 1 promoter. Above 30 °C, the rates of template association and open complex formation are both rapid ( t 1 2 ~ 10 to 15 s ), and are insensitive to moderate changes in temperature or the concentration of monovalent or divalent cations. The rate constant for open complex formation is not altered by substantial changes in enzyme or DNA concentration. Thus the rate at 30 °C is not limited by promoter melting, but possibly by formation of an early, transient promoter complex. Close comparison of the rates of template association and of open complex formation suggests that there may be an additional intermediate between the closed promoter complex and the open promoter complex. At temperatures below 25 °C, the rate of open complex formation decreases substantially, while the rate of template association remains relatively unchanged; hence, a complex accumulates having the properties of a closed promoter complex. The rate of open complex formation at 15 °C also becomes sensitive to the counterion concentration. Thus for the T7 A 1 promoter, it appears that the rate-limiting step is different at low temperatures, and involves the melting/isomerization of the closed promoter complex to the open promoter complex. Thus, even for a single promoter site, more than one step can be rate determining in the pathway from free RNA polymerase to elongating RNA polymerase, depending on the conditions employed for transcription.