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. 2016 Sep 15;63(6):939–950. doi: 10.1016/j.molcel.2016.08.011

Figure 5.

Figure 5

Region σ3.2 Is a Major Determinant of Initiation Pausing

(A) Structural model of an ITC highlighting the clash between nascent 6-nt RNA (in red) and σ3.2 (in orange). Colors as in Figure 1A.

(B) Comparison of transcription by Δ3.2 versus WT RNAP on lacCONS. Lanes 1–3: RNAs produced by Δ3.2 complexes able to synthesize up to 7-nt RNA (lane 1), up to 11-nt RNA (lane 2), and up to a run-off product (a 25-mer; lane 3). Lanes 4–6: RNAs produced by same mixtures as for lanes 1–3, but for WT RNAP.

(C) Heatmaps for Δ3.2 complexes in RPITC≤7. Right-side histogram: collapse of all E∗ values in the high-FRET plateau (reached at ∼12 s; gray bars). Frame time: 200 ms. The E∗ full width at half maximum was ∼0.34 for Δ3.2, ∼2-fold wider than for WT (0.18 ± 0.02, pink bars).

(D) Time trace where E∗∼0.45 is sampled frequently and without long pauses at E∗∼0.37.

(E) Dwell-time distributions of Δ3.2 scrunched states (n = 392).

(F) Retention of 6-nt and 7-nt RNAs in complexes due to σ3.2 presence. Reactions for RPITC<7 were run for 20 s at 37°C on bead-immobilized RPo; reactions were stopped, and complexes were washed and incubated for ∼2 min before gel loading. WT panel: using WT sigma and no washing (“T” lane), in vitro transcription yields 6-mers and 7-mers, as well as unresolved 3/4-mers. As for lacCONS, the 6-mer is more abundant than the 7-mer, consistent with pausing at 6-nt RNA. After washing and incubation (“B” lane), little 3/4-mer is retained; in contrast, there is much higher retention of 6-mer and 7-mer RNA. Lower inset: sample from lane B was run in a separate lane and overexposed. Δ3.2 panel: using Δ3.2 and no washing, in vitro transcription yields 6-mers, 7-mers, and unresolved 3/4-mers; as for lacCONS, with the 6-mer/7-mer distribution shifted substantially to 7-mer. There is little retention for 3/4-mers and 6-mer in Δ3.2, although there is moderate retention of the 7-mer, likely due to a more stable RNA-DNA hybrid. Right panel: quantitative comparison of RNA retention on bead-immobilized RPITC<7; results reflect mean and SD of four independent experiments.