Figure 3.

Effects of strengthening or weakening the σ⁷⁰ region 4/β flap interaction on λQ's engagement of the paused early elongation complex in vitro.

1. Schematic of template used for exonuclease III challenge assays. Depicted is the λQ-engaged paused elongation complex. The λP_R' template used in the assays is end-labeled at the 5′ end of the template (bottom) strand as indicated. Also indicated are the positions (relative to the transcription start site) at which the progress of exonuclease III digestion is blocked by λQ (−31) and RNAP (−22 and −12). The red DNA segment is the pause-inducing sequence; σ⁷⁰ region 2 is shown bound to the non-template strand (Ring et al., 1996). The RNAP outline is dashed to indicate that it does not depict a barrier to exonuclease digestion.
2. Exonuclease challenge assays. Stalled elongation complexes were formed with RNAP reconstituted with wild-type σ⁷⁰, σ⁷⁰ L607P, or σ⁷⁰ T544I/D581G. These complexes were then incubated with 500 nM λQ and challenged with exonuclease III for the indicated times. Lanes 9 and 18 contain A+G sequencing ladders.
3. Effects of substitutions that strengthen (T544I/D581G) or weaken (L607P) the σ⁷⁰ region 4/β flap interaction. The exonuclease barriers shown in panel B were quantified with Imagequant, and label in the λQ-dependent band at −31 was plotted as a fraction of the sum of label in the bands at −31, −22 and −12; the barriers at −22 and −12 are specific for the σ⁷⁰-dependent paused elongation complex, and are produced after exonuclease digests past the λQ barrier. We note that no differences in pause half-life were observed with these mutant holoenzymes (Nickels et al., 2005), suggesting that the effects of these σ⁷⁰ substitutions on the half-life of the λQ barrier are not indirect manifestations of changes in the stability of the paused elongation complexes.