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. 2006 Feb 16;25(5):1137–1147. doi: 10.1038/sj.emboj.7600990

Figure 5.

Figure 5

ATP-DnaA represses the nrdAB promoter more effectively than ADP-DnaA. (A) Western blot analysis. Wild-type DHB4 strain (top) or dnaAT174P mutant strain (bottom) containing an empty vector or a plasmid carrying either dnaA, dnaAA345S or dnaAT174P gene under IPTG control were grown in the presence of 100 μM of IPTG to mid exponential growth. Equal amounts of total cellular proteins were separated by SDS–15% PAGE. Subunit R2 (nrdB) of the RNR was detected by Western blotting. As loading control, the cytoplasmic thioredoxin was also detected by Western blotting. (B) In vitro transcription. Run-off in vitro transcripts from a 381 bp FokI–ClaI restriction fragment that contains promoters dnaAp1 and dnaAp2, and from a 608 bp PCR fragment that contains the nrdAB promoter and two additional uncharacterized promoters pX1 and pX2. Purified ATP- or ADP-DnaA wild-type protein (200 nM) was preincubated with DNA template for 5 min, then the RNA synthesis was started by addition of RNA polymerase (66 nM) and NTPs. Samples were separated by 6%-urea polyacrylamide gel electrophoresis. The transcripts dnaAp1, dnaAp2 and nrdAp (290, 200 and 288 bp, respectively) are indicated. Transcription from pX1 and pX2 are used as internal control for the experiments.