E. coli RNAP-catalyzed RNA extension. (A) Extension of the unlabeled (left) or 5′ endlabeled (right) RNA oligonucleotide LR-3. The right panel is pseudocolored to reflect the 5′ 6-FAM endlabel. Oligonucleotide sequence is an arbitrary segment of 68 nt from the coding region of GFP (sequence in Supplemental Table S1). In the left panel, the gel was stained with SybrGold and imaged. In the right panel, the fluorophore was imaged (to visualize the 5′ 6-FAM); the gel was then stained with SybrGold and reimaged (to visualize the ladder). Extension was overnight (∼12 h). (B) Time course of RNA extension with the unlabeled LR-3 RNA oligonucleotide. (C) Schematic of proposed model for RNA extension. The LR-3 RNA oligonucleotide is in black, the RNAP in blue, and the additional nucleotides in red. Note that the diagram is not to scale.
