Figure 4.

Substitutions in the σ⁷⁰ NCR enhance pausing at λP_R′ and at placUV5. (A) Single-round in vitro transcription time-course assays using a P_R′ template (left and middle panels) or a placUV5 template (right panel) and RNAP reconstituted with the indicated σ⁷⁰ proteins. Aliquots of single reactions were removed and stopped at the indicated time points after transcription was initiated. The RNA was labeled internally with [α-³²P]UTP (middle and right panels) or end-labeled with [γ-³²P]ATP (left panel). +16 and +17, 16- and 17-nt RNA species, respectively, produced from the λP_R′ template; T, 194-nt terminated transcript produced from the λP_R′ template; +17 and +18, 17- and 18-nt RNA species, respectively, produced from the placUV5 template; RO, 96-nt runoff transcript produced from the placUV5 template. A faint 18-nt RNA species was also observed during time courses with the λP_R′ template when the RNA was internally labeled. This is likely the result of nucleotide deprivation at U19 under conditions where the UTP concentration was reduced to improve incorporation of [α-³²P]UTP, and has been observed previously under similar reaction conditions (Ko et al, 1998). (B, C) Effects of substitutions on pause capture and pause half-life. The percentage of elongation complexes paused (100 (16-nt+17-nt)/(16-nt+17-nt+T) or 100 (17-nt+18-nt)/(17-nt+18-nt+T)) was approximated at each time point, plotted, and fit to the exponential equation Y=Y₀e^−kt (Supplementary Figure S1). Exponential equations were solved to obtain pause capture (left panels) and half-life (right panels) values for each holoenzyme. Pause capture was approximated by extrapolating the equations to t=0. Error bars represent standard deviations from at least three separate experiments. In cases where error bars are not shown, the mutants were assayed twice, with similar results.