FIGURE 5.

Fractional occupancies of E. coli σ⁷⁰-R2 and σ⁷⁰-R4 in EC on lacUV5 promoter. A, representative data assessing the components of RP_o and EC. RP_o and ECs were formed as described in the legend to Fig. 2A. The beads containing RP_o and EC were washed before resolving on a 4–20% gradient SDS-PAGE, followed by fluorescence scanning and Coomassie staining. B, fractional occupancy of σ⁷⁰-R2 and σ⁷⁰-R4 with respect to RNAP core: determined by quantifying the amount of each protein from the gel. Data were the average of six replicates. Gray bar, σ⁷⁰-R2; black bar, σ⁷⁰-R4. C, representative data for in vitro transcription assay with RP_o immobilized on streptavidin beads. Stalled elongation complexes EC+15, EC+25, and EC+40 were generated using ³²P-labeled NTP; chase, all four NTP were added to EC to produce runoff products. The samples were run on 12% urea-PAGE and scanned on phosphorimager. D, fractional occupancies of σ⁷⁰-R2 and σ⁷⁰-R4 after correction for the subpopulation that are competent to form EC. Data were average of six replicates. Gray bar, σ⁷⁰-R2; black bar, σ⁷⁰-R4.