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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 4.

Figure 4

82Q can engage a transcription initiation complex.
  1. Left panel shows a diagram of the wild-type phage 82 PR' promoter. Indicated are the functionally important elements at phage 82 PR' including the promoter −10 and −35 elements, the 82Q-binding element (QBE), the pause-inducing extended −10-like element (located between +6 and +14), and terminator t82. Right panel shows a diagram of the modified template on which the 82 PR' promoter was inactivated with base pair substitutions in the promoter −10 and −35 elements and the pause-inducing element was converted to a consensus extended −10 promoter element. The transcription start site on each template is indicated (bent arrow).
  2. Effects of strengthening (T544I/D581G) or weakening (L607P) the σ70 region 4/β flap interaction on 82Q's engagement of a transcription initiation complex. Shown are the results of representative single-round in vitro transcription assays performed in the presence or absence of 100 nM 82Q using the indicated template and holoenzyme reconstituted with wild-type σ70, σ70 T544I/D581G, or σ70 L607P. The percentage of transcripts derived from terminator readthrough (readthrough/ [readthrough + terminated]) is indicated above each lane. RNA was end labeled using γ-32P-ATP (lanes 1 and 2) or γ-32P-GTP (lanes 3-12). Also indicated are the 81- (lanes 1 and 2) or 62- (lanes 3-12) nt terminated transcript (T) and the 116- (lanes 1 and 2) or 97- (lanes 3-12) nt readthrough transcript (RT). Plotted in the bar graph on the right are the averages of 3-6 independent measurements (and standard deviations).
  3. Effect of removing σ70 region 4 on 82Q's engagement of a transcription initiation complex. Shown are the results of representative single-round in vitro transcription assays performed in the presence or absence of 100 nM 82Q using the modified phage 82 PR' template and holoenzyme reconstituted with wild-type σ70, σ70 L607P, or σ70 lacking region 4 (σ70 1-529). The percentage of transcripts derived from terminator readthrough (readthrough/[readthrough + terminated]) is indicated above each lane. RNA was internally labeled using α-32P-CTP. Also indicated are the 62-nt terminated transcript (T) and the 97-nt readthrough transcript (RT). Plotted in the bar graph on the right are the averages of 4-6 independent measurements (and standard deviations).