Figure 2: Effects of PPi on Synthesis of Long RNA at 37 °C.

Panel A) The nontemplate-strand sequence of the core region of the modified λP_R promoter (in a 122 bp (−80 to −42) promoter fragment) used in this and previous studies of step-by-step initiation kinetics [12]. Panel B) Single-nucleotide resolution PAGE separations of ³²P-labelled RNA products at different times in initiation (10–480 s after addition of 200 μM ATP, 200 μM UTP, 10 μM GTP, 17.5 nM α-³²P-GTP to preformed OC) in manual-mixing experiments at 0 mM and 2mM PPi. Omission of CTP causes a stop at 16-mer, with subsequent readthrough to a second stop at 31-mer. Different run-times were used for the two gels. The partial lane at left is from an unrelated experiment. Panel C) Time courses of synthesis of long RNA (11-mer or longer, designated as 11+) in single-round experiments at ≤ 3 mM PPi at 37 °C. Amounts of 11+ RNA are normalized by the total amount of OC. Single exponential fits are shown as solid curves. Error bars show one standard deviation from the average of normalized data (see Methods) from 3–4 experiments. Panel D) Half-times t_1/2 (t_1/2 = 0.69/k₁₁₊) for synthesis of 11+ RNA (black) and amounts of 11+ RNA synthesized (red) are plotted for each PPi concentration at 37 °C. Experimental uncertainties are approximately ±20% for all points. Without added PPi, t_1/2 is too small to determine by manual mixing. Fast-mixing experiments at 37 °C gave t_1/2 ≈ 1.8 s (k₁₁₊ 0.4 s⁻¹) [12]. Dashed lines are included as a visual guide.