Fig. 3.
Enzymatic test for assessing the binding of T7 RNAP to the promoter. (A) Position of AseI sites in the plasmid shown in Fig. 2A (g10 ITS). The PT7 promoter used here (enlarged) encompasses an AseI site (underlined; cleavage is shown by arrow) that can be shielded by T7 RNAP binding (see B and C). The ITS is italicized; the nucleotides that are not strictly conserved among T7 late promoters are in small capitals. Before the assay, the plasmid was linearized with DraIII (see text). The size of the AseI and AseI-DraIII fragments is indicated. (B-C′) AseI digest of the plasmid shown in A in the presence of no RNAP or WT or P266L T7 RNAP, as indicated above each lane. The disappearance of fragments I and III and the accumulation of the uncut fragment I+III are diagnostic of promoter protection. The assay has been done without NTPs (-) or in the presence of either GTP (G), or GTP + ATP (G,A), which allow transcription to nucleotides +3 and +6, respectively. B and C correspond to low- and high-salt conditions, respectively; C′ is similar to C, except that AseI digestion was slightly longer, making variations in the intensity of the I+III band more visible.