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. 2015 Jan 16;4:e04970. doi: 10.7554/eLife.04970

Figure 7. Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination.

(A, B) Densitometry profiles of transcripts produced at 28°C, 8 mM Mg2+, and 30 µM each NTP from the λPR-bgl template in bridged filaments (66 H-NS/kb) with or without 5 nM Rho (A) and with or without 75 nM NusG (B). Samples were removed at 2, 4, 8, 16, and 32 min after NTPs were added and separated by denaturing PAGE. To detect release of Rho-terminated transcripts, a 32-min sample was separated into released and EC-bound fractions using paramagnetic Co2+ beads that bind the His10-tagged RNAP. The released supernatant fraction was separated by denaturing PAGE and converted to densitometric profiles shown as darker colors. (C, D) Mean transcript lengths and standard deviations were calculated from at least two (C) or four (D) independent experiments. (E) Rho termination efficiencies were calculated as the fraction of released transcripts divided by the total transcripts, with averages and standard deviations from at least three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.04970.021

Figure 7.

Figure 7—figure supplement 1. Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination.

Figure 7—figure supplement 1.

(A) Halted A26 ECs (10 nM) formed on the λPR-bgl template were incubated with bridged H-NS, 75 nM NusG, or 5 nM Rho at 8 mM Mg2+ at 28°C. Samples were then collected at 2, 4, 8, 16, and 32 min after addition of NTPs (30 µM each) and resolved by PAGE. Rho-terminated, released transcripts for the 32-min sample were determined as described in the legend to Figure 7. R, released transcripts. M, labeled MspI-digested pBR322 marker. RO, run-off RNAs. (B) Densitometry profiles were determined as described in legend of Figure 2. Mean transcript lengths were averaged from at least two independent experiments. (C) Mean transcript lengths and standard deviations with or without bridged H-NS and with or without 5 nM Rho were determined using at least four independent experiments. (D) Mean transcript lengths with or without bridged H-NS, 5 nM Rho, or 75 nM NusG were averaged from two independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.04970.022