Figure 3.

σ⁷⁰ fragments used and their binding to RNAP core. (A) A scheme of σ⁷⁰; the universally conserved regions are shown in white and are numbered. The structural domains of σ (σ1.1, σ2, σ3, and σ4; Campbell et al, 2002) are indicated beneath. σ fragments used in this work are shown as simple lines below the σ⁷⁰ scheme. Contact sites with the −10 and −35 promoter elements and the β′ coiled-coil (β′c–c) are indicated. (B, D) Inhibition of σ⁷⁰-dependent abortive transcription initiation reaction from the lacUV5 promoter by σ⁷⁰ fragments. Concentrations of σ⁷⁰ and its fragments are indicated at the top of the panel. Reactions contained either 50 nM (panel B) or 10 nM (panel D) of RNAP core. ³²P-labeled abortive ApUpU RNA product is marked. (C) Quantification of experimental data presented in panel B. The amount of ³²P-labeled RNA synthesized in the presence of σ fragments was normalized to the amount synthesized without fragments added (black bar at the left). Gray bars correspond to lanes 2, 5, 8, and 11 of panel B. Light gray bars correspond to lanes 3, 6, 9, and 12. White bars correspond to lanes 4, 7, 10, and 13. Mean values and s.d. from two independent experiments are shown. (E) Quantification of experimental data presented in panel D. The amount of ³²P-labeled abortive product synthesized in the presence of indicated concentrations of σ fragments is shown.