FIG. 2.

EMSA of Pmga cre using His-CcpA. (A) Sequence of annealed oligonucleotide (Oligo) probes used in EMSAs. Putative cre are indicated by shaded boxes. Base pairs matching Pmga cre are shaded and in boldface. EMSA was performed on radiolabeled PccpA-annealed (B) or (C) Pmga-annealed oligonucleotide probes. A constant amount (1 to 2 ng) of [γ-³²P]ATP-labeled probe was incubated with increasing amounts (5 to 12.5 μM) of purified GAS His-CcpA for 30 min at 30°C prior to separation on a 5% polyacrylamide, 10% glycerol gel. The specificity of His-CcpA binding to Pmga was assayed by addition of 700 ng of unlabeled competitor annealed oligonucleotide probes corresponding to Pmga (lane 6), mutated (Mut.) Pmga (lane 7), PccpA (lanes 4 and 8), and scrambled (Scr.) PccpA (lane 5) or Pmga (lane 9) using the oligonucleotide pairs listed in Table 2.