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. 2002 Nov 1;30(21):4658–4666. doi: 10.1093/nar/gkf605

Figure 5.

Figure 5

In vitro psbA2 basal transcription and factors contributing to the transcription. (A) In vitro mRNA synthesis by two different RNAPs from the mutagenized CIT promoter. Using the RNAP of Ec + σA1 (1 pmol purified E.coli core enzyme + 3 pmol K-81 principal sigma factor σA1) or *EσA1 [partially purified RNAP fraction containing K-81 σA1; 15 µl (∼2 pmol RNAP)], in vitro mRNA synthesis was carried out from the DNA template pAG431 or pAD501 (3 µg). The synthesized mRNA was examined by in vitro primer extension with the primer lacZ-R1 (7). Relative values of synthesized mRNA (%) according to the signal intensity on X-ray film are shown. (B) DNA binding of RNAP components to the psbA2 upstream region. (Top) A schematic representation of template DNAs. A 576 bp EcoRI (E)–HindIII (H) fragment (Fig. 1) isolated from pHNL7-up (the –404 to +113 psbA2 region) (7) was digested with the respective restriction enzymes (see Fig. 1). These fragments were end-labeled, then the mixture was subjected to the assay. DNA fragments (1 µl, 1 pmol) were mixed with (left) K-81 *EσA1 (0.25 or 1 µl RNAP at 0.4 pmol/µl) or (right) Ec + σA1 (0.25 or 1 µl at 0.4 pmol/µl). The gel profile for no addition of protein is indicated as –. The positions of the DNA fragments, DNA–protein complexes (Cs) and gel origin (Ori) on the gel are shown.