FIG. 3.

Core RNAP binding analysis of ς³² proteins by glycerol gradient sedimentation. Equimolar concentrations of core RNAP and different ς³² proteins (final concentration of 100 nM) were allowed to reconstitute at 30°C for 15 min in a buffer containing 50 mM HEPES (pH 7.9) at 4°C, 0.1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 10 mM MgCl₂. The mixture was loaded onto the top of a 5-ml 15 to 35% glycerol gradient and centrifuged at 48,000 rpm for 24 h at 4°C in a Beckman SW50.1 rotor. Sixteen fractions were collected from the bottom of the tube and subjected to immunoblot analysis. The sedimentation patterns of each ς³² protein were detected with anti-ς³² serum. The positions of β and β′ subunits were determined with polyclonal antibodies to core RNAP. The leftmost region of each panel represents the bottom of the tube (35% glycerol). The positions of core RNAP were indistinguishable with (A) or without (data not shown) ς³². The sedimentation patterns are shown for ς³² only (B), for two different wild-type proteins with core RNAP (C and D), and (as indicated above each panel) for the mutant proteins with core RNAP (E through M).