FIG. 6.

Multiple rounds of transcription in vitro of the leuV(−50 +11) promoter as a function of NTP concentration in the presence of 200 mM KCl. (A) Autoradiogram of 5% denaturing polyacrylamide gel representing the separation of the transcription products in the presence of a fixed concentration of GTP (200 μM) and increasing concentrations of CTP. The concentrations of CTP are 3, 8, 24, 72, 216, 670, 2,000, and 6,000 μM (lanes 1 to 8, respectively). The template employed contained leuV promoter sequences from positions −50 to +31, which produced 86-nucleotide transcripts. (B) Nucleotide dependence of the leuV promoter in multiple rounds of transcription. Increasing concentrations of ATP (filled triangles), CTP (open circles), GTP (filled circles) and UTP (open triangles) are shown. In the case of ATP and UTP titrations, two bands corresponding to initiation at C7 and G9 were seen at all concentrations. In this case, values plotted are the sum of these two transcripts. In the case of CTP, only counts corresponding to initiation at C7 were quantitated and plotted. Similarly, in the case of GTP titrations, only counts in G9 were quantitated and plotted. (C) Initiating nucleotide dependence of promoters in multiple rounds of transcription. Transcriptional activities as a function of initiating nucleotide concentrations are presented for leuVp (circles), tacp (squares), and rrnBp₁ (triangles). Multiple rounds of transcription were performed in the presence of 200 mM KCl as described in Materials and Methods. In the case of leuV and tac promoters, various concentrations of GTP were employed. For rrnBp₁, increasing ATP concentrations were used.