Fig. 5.

Analysis of kinetic parameters of RNAP-promoter complex formation. A) Multiple steps in the process of forming an RNAP-promoter open complex. R: (RNAP); P: promoter DNA; RP_c: closed complex, competitor-sensitive; RP_I: intermediate complex, DNA strands closed; RP_o: open complex, DNA strands open. RP_e: elongation complex. NTP: nucleotide substrates. RP_o is a competitor (heparin) resistant complex at many, but not all, promoters. K_B (or K₁): equilibrium constant for formation of RP_c; k_a: composite second order association rate constant; k_f (or k_i): RNAP concentration-independent isomerization rate constant (may reflect rates for multiple intermediate steps at some promoters); k_d: composite dissociation rate constant. (see [1,2,52]). (B) Time course for formation of a heparin resistant RNAP complex at the lacUV5 promoter. The ³²P end-labeled fragment containing the E. coli lacUV5 promoter (−130 to +40) was incubated with 5 nM RNAP at 30°C in buffer containing 110 mM KCl, 10 mM Tris-Cl pH 8.0, 10 mM MgCl₂, 1 mM DTT, 100 μg/ml bovine serum albumin (BSA). Aliquots were removed at time intervals, heparin was added (to 10 μg/ml final), and samples were filtered through nitrocellulose filters [86]. The rate constant, k_obs, for formation of the complex at this RNAP concentration (7 × 10⁻³ s⁻¹) was determined from Eq. 1, as described in text. (C) Determination of the dissociation rate of an RNAP-lacUV5 promoter complex. Complexes were formed with a ³²P end-labeled lacUV5 promoter fragment (−130 to +40) and 10 nM RNAP at 30°C in the buffer described for panel (B). Competitor (heparin, final concentration 10 μg/ml) was added at t=0, and aliquots were filtered at time intervals. A semi-log plot of complexes remaining as a function of time (in minutes) is shown. The dissociation rate (k_d) determined from a single exponential fit of the data was 2.3 × 10⁻⁴ (s⁻¹). (D) Tau plot (1/k_obs vs 1/[RNAP]) of observed rate constants, k_obs, for formation of a heparin resistant RNAP-promoter complex (RP_AC) with a ³²P end-labeled fragment containing the E. coli rrnB P1 promoter (−88 to +50), determined at a series of RNAP concentrations. Heparin (10 μg/ml) stable complexes at rrnB P1 are formed in the presence of the first two NTP substrates (ATP, CTP), which produces a 5-mer transcript [58,63]. Complexes were formed at 25°C in buffer containing 45 mM NaCl, 10 mM Tris-Cl pH 8.0, 10 mM MgCl2, 1 mM DTT, 100 μg/ml bovine serum albumin (BSA), 500 μM ATP and 50μM CTP. Values for k_obs were determined as shown in panel B, using Eq. (1). The overall association rate constant, k_a, determined from a fit of the data to Eq. (2), was ~3.0 × 10⁷ M⁻¹ s⁻¹, and k_f was 1.2 × 10⁻² s⁻¹. (E) Nonlinear plot (k_obs vs. [RNAP]) of the same data as in (D). k_a, from fit of data to Eq. (3) was 3.3 × 10⁷ M⁻¹ s⁻¹, and k_f was 1.2 × 10⁻² s⁻¹.