FIGURE 4.

RNAP containing σ⁷⁰F610A or σ⁷⁰Δ604–613 is more efficient at promoter clearance. A, sequences of the −35/−10 promoter P_uvsX/σ* and the extended −10 promoter galP1/cons. B and C, single round in vitro transcription using WT and the indicated mutant σ and either P_uvsX/σ* (B) or galP1/cons (C). The labeled nucleotide in the reaction was [α-³²P]ATP. Each template produces a full-length product of 34 nucleotides as well as abortive RNAs from 2 to ∼10 nucleotides. Because the RNA products were treated with phosphatase before electrophoresis, the abortive RNAs migrate according to size and base composition (see supplemental Fig. S2 for details). Histograms below show the amount of abortive RNA relative to the amount of full-length RNA for each lane. In the case of galP1/cons, the amount of short RNAs made using RNAP lacking σ was also subtracted as background before determining the relative ratio. The mutations having the most significant effect, F610A and Δ604–613, are shown in red. S.D. values (error bars) were determined from three or more independent experiments. The error bars with RNAP containing σ⁷⁰D612A are too small to be seen.