Fig. 3. Effect of the GalR mutant R282L on the transcription of the P1 and P2 promoters in vitro. (A) Schematic drawing of the gal regulatory region in plasmid pSA850 used as a template DNA in the in vitro transcription assay. The wild-type regulatory region, containing the external (OE) and the internal (OI) operator sites and the P1 and P2 promoters, is followed by the rpoC terminator. Transcription termination at the rpoC terminator results in a 125 nucleotide RNA from P1 and a 130 nucleotide RNA from P2. The numbering system used the +1 start site of P1 as reference. The HU-binding site (hbs) is centered at position 6.5. (B) In vitro transcription assay performed on supercoiled pSA850 DNA in the presence of wild-type (left panel) and R282L mutant (right panel) GalR protein. The RNA product was resolved on 8% polyacrylamide gels. The concentration of GalR and HU was 80 nM as shown on the top. The 80 nucleotide RNA1 transcript, which is independent of HU and GalR concentrations, served as an internal control between lanes. (C) Effect of operator deletions on the transcription of the gal promoters. Transcription on supercoiled pSA886 (OEOIN) DNA (left panel) and pSA887 (OENOI) DNA (right panel) in the presence of the wild-type and R282L mutant GalR protein. The concentration of the repressor proteins was 80 nM, as indicated on the top. Transcripts, marked P1 and P2, are RNAs made from the gal promoters. The transcript, marked RNA1, serves as an internal control. (D) Transcription of the gal promoters on linear pSA850 DNA. pSA850 was treated with the restriction enzyme HindIII. The HindIII restriction site is located 491 bp downstream of the OI operator site. The total length of the plasmid is 3.5 kb. Transcription was performed as described in Materials and methods. Proteins were used at the concentrations indicated on the top. Transcripts, marked P1 and P2, are RNAs made from the gal promoters. The transcript, marked RNA1, served as an internal control.