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. 2007 Feb 1;26(4):955–964. doi: 10.1038/sj.emboj.7601555

Figure 4.

Figure 4

Binding of the exogenously added σ70 fragments to paused TEC16. (A) Crosslinking of TEC16 complexes to 32P-labeled lacUV5 DNA before (lane 1) and after (lanes 2–7) heparin wash. Heparin-washed σ70-less complexes were supplemented with 0.5 μM of wild-type σ70 (FL) or fragments: 5 μM of σ2a, 3 μM of σ2−3, 1.5 μM of σ1−2, and 2.5 μM of σ2.4 (lanes 3–7). Crosslinked complexes were resolved by SDS–PAGE and revealed by autoradiography. Crosslinked subunits are indicated. (B) Crosslinking of TEC16 complexes washed with heparin (lanes 2–9) and supplemented with 1.5 μM of σ2b or σ1−2 (lanes 3–6, 8, and 9). NTPs were added before (lanes 7–9) or after (lanes 5 and 6) the addition of σ70 fragments. Lane 1: TEC16 before heparin wash. Reaction products were analyzed as in panel A. (C) 32P-labeled RNA transcripts produced upon addition of NTPs to immobilized TEC16. Complexes were chased by the addition of NTPs for 0.5, 1, and 5 min. Lanes 5–19: complexes were supplemented with wild-type σ70 (FL) or indicated σ70 fragments before addition of NTPs. (D) Quantification of the results of experiment shown in panel C. The amount of +17 RNA was calculated as percentage of the initial amount of the starting 16-mer RNA present before the addition of NTPs. Mean values and s.d. from two independent experiments are shown.