Fig. 4. Supershifting of the PacC(5–430)–DNA complex by purified GST::PacC fusion proteins containing the C–terminus of PacC. Acidic grown strains were used. (A) The indicated GST::PacC proteins were added to binding reactions containing a pacC^c75 (truncating PacC after residue 430) extract and the ³²P-labelled ipnA2 PacC binding site, and the resulting complexes were resolved by electrophoresis in an 8% (w/v) polyacrylamide gel. With no addition or upon addition of GST, a major complex corresponding to the processed PacC form (open arrow, whose predominance is favoured by the alkalinity-mimicking, truncating pacC^c75 mutation) and a minor complex corresponding to the PacC(5–430) translation product (solid arrow) are resolved. The latter is supershifted by all GST fusion proteins containing PacC residues 529–678. A wild-type extract (acidic growth conditions) was used in lane 8 as a control. (B) Antiserum raised against a GST::PacC(529–678) fusion protein prevents supershifting, whereas anti-GST antiserum prevents penetration of the supershifted complex into the gel. Only the relevant portion of the gel is shown. (C) Certain alkalinity-mimicking single-residue substitutions within residues 529–678 prevent supershifting.