Figure 5.

Evaluation of magnesium and manganese requirement on RNA polymerase activity. RNA products generated from in vitro transcription assays containing various concentrations of magnesium and manganese metal ions were separated by gel electrophoresis in a 10% TBE-Urea gel and detected using a phosphor screen. Representative gels are shown from in vitro transcription reactions prepared in parallel with metal salts: reactions containing (A) 0–20 mM magnesium or (B) 0–20 mM manganese or (C) 2 mM magnesium with 0–20 mM manganese. Data are representative of three replicate experiments and gels were cropped from a single image. Phosphor screen signals were quantified by densitometry and plotted over varied magnesium or manganese concentrations (D). Differences in signal intensity achieved using manganese alone (1–20 mM Mn²⁺) and those supplemented with magnesium (1–20 mM Mn²⁺ with 2 mM Mg²⁺) were analyzed by a two tailed paired t-test matched along the entire metal concentration range and was found to be statistically significant (α = 0.05, p = 0.004), indicating a role for magnesium in RNAP activity.