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. Author manuscript; available in PMC: 2007 Nov 3.
Published in final edited form as: Mol Cell. 2006 Nov 3;24(3):457–468. doi: 10.1016/j.molcel.2006.09.014

Figure 2.

Figure 2

Effects of strengthening or weakening the σ region 4/β flap interaction on λQ-mediated antitermination
  • A) and B) Effects of strengthening (T544I/D581G) or weakening (L607P) the σ70 region 4/β flap interaction in vitro. Shown are the results of single-round in vitro transcription assays performed using a linear template that contains sequence extending from −109 to +238 of λPR' that includes the natural terminator tR' (Figure 1A). Assays were done either at a low concentration of λQ (2.5 nM, panel A) or at a high concentration of λQ (500 nM, panel B). Graphs show the percentage of transcripts derived from terminator readthrough (readthrough/[readthrough + terminated]) at the indicated times after transcription was initiated. Reactions were performed using holoenzyme reconstituted with wild-type σ70, σ70 L607P or σ70 T544I/D581G, as indicated. Assays were performed three times on separate occasions with similar results. Plotted on the graphs are the data obtained from a single representative experiment.
  • C) Effects of weakening (F278L) the σ38 region 4/β flap interaction in vitro. Shown are the results of single-round in vitro transcription assays performed at a low concentration of λQ (10 nM). Graphs show the percentage of transcripts derived from terminator readthrough (readthrough/[readthrough + terminated]) at the indicated times after transcription was initiated. Reactions were performed using holoenzyme reconstituted with either wild-type σ38 or σ38 F278L, as indicated. Assays were performed three times on separate occasions with similar results. Shown on the graph are the data obtained from a single representative experiment.
  • D) Effects of strengthening (T544I/D581G) the σ70 region 4/β flap interaction in vivo. Reporter strain cells containing either wild-type σ70 or σ70 T544I/D581G and harboring a λPR'-lacZ reporter were transformed with a plasmid that did or did not encode λQ. The cells were grown in the presence of 100 μM IPTG and assayed for β-galactosidase activity. The bar graph shows the averages of four independent measurements (and standard deviations).