Figure 5. Effect of 8-amino-ATP on in vitro transcription.
A. Schema of Assay. Step 1: The AFP7 plasmid was linearized by restriction enzyme digest and dephosphorylated. Step 2: In vitro transcription reaction was performed using α-32P-UTP to label nascent RNA. Step 3: Nascent RNA was purified and separated by gel electrophoresis. B. Effect of various concentrations of 8-amino-ATP and ATP on in vitro transcription of 250 nucleotide RNA product. Experiments were performed as described in schema. C. Quantitation and normalization of the in vitro transcription reactions with 0.6 μM ATP and various concentrations of 8-amino-ATP or the known RNA synthesis inhibitor α-amanitin. D. Quantitation and normalization of the reactions with 0.6 or 2.4 μM ATP with or without an equal concentration of 8-amino-ATP.