Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of binding at bacteriophage T7 promoter A1 using a template competition assay

Abstract We have studied the association of Escherichia coli RNA polymerase holoenzyme with DNAs from two deletion mutants of bacteriophage T7 that contain only the strong A1 promoter using a template competition procedure. The DNA templates employed each bear a transcription unit of a distinct size read from T7 promoter A1. Association of holoenzyme with one template is followed by adding an excess of the second template to interrupt binding. The distribution of enzyme on each DNA is then determined in a second reaction, in which each enzyme initiates transcription from the A1 promoter and is allowed to complete a single RNA chain. The number of chains read from the A1 promoter of each template is analyzed by quantitative gel electrophoresis. The procedure can be used to measure both association and dissociation rates, as well as binding of holoenzyme to promoter sites of a template at equilibrium. Only enzymatically active holoenzyme is detected, and association can be measured under conditions such as low temperature or elevated salt concentrations, where open promoter complexes are not stable. The rate of association between E. coli RNA polymerase holoenzyme and the T7 A1 promoter measured by template competition is rapid at 37 °C in solutions containing 15 m m -NaCl, 10 m m -MgCl2 at pH 8 ( t 1 2 ~ 10 s ), and slightly faster than the rate of open promoter complex formation ( t 1 2 ~ 15 s ) (Rosenberg et al., 1982). The rate is proportional to enzyme concentration, but is not altered over at least a twofold range of DNA template concentrations. The rate is not highly sensitive to the concentration of NaCl, nor to temperature over the range from 37 °C to 15 °C. We conclude that the rate of association at 37 °C is limited by an apparent first-order process in which DNA melting is not involved. Studies of other promoters suggest that RNA polymerase can locate a promoter much more rapidly than the rate of association we measure. Hence, we suggest that the rate-limiting step at 30 °C for the T7 A1 promoter may be conversion of a specific, but transient, initial promoter complex to the more stable closed promoter complex. Since the rate of open promoter complex formation drops considerably at lower temperature (Rosenberg et al., 1982), RNA polymerase becomes associated with the DNA template much more rapidly than it forms open promoter complexes at these temperatures (below 20 °C). The kinetic intermediate defined by this result has the properties expected of a closed promoter complex. Equilibrium studies at 0 °C show that RNA polymerase associates with the T7 A1 promoter with an association constant Ka = 4.4 × 109 m−1. This complex has a dissociation rate of 2.9 × 10−2 s−1 ( t 1 2 ~ 24 s ), and is therefore much less stable than previously suggested. Equilibrium studies at 37 °C in solutions containing 10 m m -MgCl2 and up to 100 m m -NaCl (pH 8) show that the active RNA polymerase added is bound almost quantitatively to the T7 A1 promoter under conditions of promoter excess. We calculate a binding constant of 6 × 1011 m−1 for binding of holoenzyme to the A1 promoter in 100 m m -NaCl, 10 m m -MgCl2 at 37 °C. The promoter binding constant is highly sensitive to NaCl concentration: a tenfold increase in NaCl concentration reduces the value of log K for holoenzyme-promoter binding by about 12.5. The equilibrium specificity ratio of RNA polymerase bound to the T7 A1 promoter relative to that free or non-specifically bound is very high, being 6 × 107 in 100 m m -NaCl, 10 m m -MgCl2.