| Method |
Researchers used the abortive initiation assay to monitor in vitro
the lags in the formation of open complexes on the two promoters, wildtype pR and mutant x3, when the reaction was initiated with RNA polymerase.
The average time necessary for open complex formation, 'Tau obs'
was measured at different RNA polymerase concentrations.
These data were analyzed in a way that allowed the equilibrium
constant for the initial binding (KI = k1/k-1) to be quantitated
separately from the rate of open complex formation (k2). This
latter process is referred to as an isomerization because it is
not known whether the rate of DNA melting
or another rate-determining conformational change was measured. Values for the kinetic constants k2 and kon were obtained from the
intercepts (intercept = 1/k2) and the slopes (1/S = k1×k2/k-1=kon)
of Fig. 2 (graph of tau observed vs. 1/[RNAP]) |