Figure 6.

CRP binds to the sdhC promoter and activates sdhCDAB expression in vitro in the presence of cAMP. (A) CRP binds to its target sites on the ptsG and sdhC promoters. ³²P-labeled DNA probes containing the ptsG or sdhC promoter regions were mixed with the indicated CRP concentrations in the presence of 100 µM cAMP and then electrophoresed on 6% polyacrylamide gels. (B) The effect of CRP·cAMP and ArcA on sdhCDAB transcription in vitro. The supercoiled DNA template, pTSDHpro, was used for the in vitro transcription. The templates were preincubated with RNA polymerase and CRP and/or ArcA as described under ‘Materials and Methods.’ The reaction was started and stopped by the addition of NTP solution containing heparin and loading dye, respectively, and analyzed on a 5% polyacrylamide gel containing 8 M urea. Lanes 2–5 contain 5, 10, 20 and 40 nM CRP in the reaction, respectively; lanes 6–8 contain 100, 200 and 400 nM ArcA with 40 nM CRP in the reaction, respectively. The transcripts from the plasmid origin of replication (106/107 nt) are marked as rep. The 248 nt transcript from sdhC promoter is indicated.