NTP requirements for RNA synthesis by Δ21 from linear and circular templates. (A) Labeling of the products from linear and circular templates with [α-32P]CTP and [α-32P]ATP. The conditions used in each reaction are indicated above the gel image. The final concentration of the radiolabel was 250 nM. GTP is present at 0.2 mM in all of the reactions, and NTPs that are not used as radiolabel are present at 0.1 mM. (B) Titration of nonradiolabled CTP into reactions radiolabeled with [α-32P]CTP (250 nM final concentration). The templates used for RNA synthesis by Δ21 and the concentrations of the nonradiolabeled CTP added to the reactions are shown above the gel image. The decrease in the signals is due to decreased specificity of labeling in the presence of nonradiolabeled CTP. The amount of 13-nt product was normalized to the reaction not amended with cold CTP. (C) Effect of increasing salt on RNA synthesis from +1C-L and +1C-C. RNA synthesis was performed with increasing concentrations of NaCl (up to 100 mM), and the 13-nt product obtained was quantified and plotted against NaCl concentration.
