FIGURE 2.
Fractional occupancies of YvrI and YvrHa of B. subtilis in EC on PoxdC promoter. A, representative data assessing the components of RPo and EC. RPo were formed by incubating 200 nm B, subtilis RNAP core, 500 nm each of TMR-labeled YvrI and YvrHa, and 100 nm biotinylated promoter PoxdC 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. B, fractional occupancy of YvrI and YvrHa with respect to the RNAP core: determined by quantifying the amount of each protein from the gel. Data were average of six replicates. Gray bar, YvrI; black bar, YvrHa. C, representative data for in vitro transcription assay with RPo immobilized on streptavidin beads. Stalled elongation complexes EC+11, EC+20, and EC+39 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. D, fractional occupancies of YvrI and YvrHa after correction for the subpopulation that are competent to form EC. Data were average of six replicates. Gray bar, YvrI; black bar, YvrHa.