FIGURE 1.
Fractional occupancy of σ70 of E. coli in EC on T7A1 and lacUV5 promoters. A, representative data assessing the components of RPo and EC. RPo were formed by incubating 200 nm Ec RNAP core, 500 nm each of TMR σ70, and 100 nm biotinylated promoter DNA fragment at 37 °C for 20 min, and immobilized on streptavidin beads. ECs were formed by adding NTP (indicated under ”Materials and Methods“). The beads containing RPo and EC were washed before resolving on 10% SDS-PAGE, followed by fluorescence scanning and Coomassie staining. Left panel, data for T7A1 promoter: EC+23 were formed. Right panel, data for lacUV5 promoter: EC+15 were formed. B, fractional occupancies of σ70 with respect to RNAP core: determined by quantifying the amount of each protein from the gel. Data were average of six replicates. Left panel, data for T7A1 promoter: EC+23 were formed. Right panel: data for lacUV5 promoter: EC+15 were formed. C, representative data for in vitro transcription assay with RPo immobilized on streptavidin beads. Stalled elongation complexes 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 a phosphorimager. Left panel, data for T7A1 promoter: EC+23 were formed. Right panel, data for the lacUV5 promoter: EC+15 were formed. D, fractional occupancies of σ70 after correction for the subpopulation that are competent to form EC. Data were average of six replicates. Left panel, data for T7A1 promoter: EC+23 were formed. Right panel, data for lacUV5 promoter: EC+15 were formed.