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. 2004 Oct 8;383(Pt 2):371–382. doi: 10.1042/BJ20040792

Figure 3. The −35 sequence is required for gapA P1 activity.

Figure 3

(a) Sequences of the variant gapA P1 promoters produced to test for the importance of the −10 and −35 sequences are shown. Positions of base substitutions are underlined. (b) Effect of absence of the −35 sequence on the in vivo level of gapA mRNAs. The RNA extracts were prepared and analysed as described in the legend to Figure 1. (c1) Plasmid pRLG::gapAP1 carrying the WT gapA P1 promoter and its derivatives carrying the mutated promoters were used as templates for the in vitro run-off transcriptions [28]. After transcription, the products were fractionated on a 5% polyacrylamide denaturing gel as described in the Experimental section. The RNA I transcript, encoded by the plasmid, was used for the normalization of the amount of gapAP1 transcript recovered in each transcription assay. (c2) For each variant promoter, the gapA P1-rrnB transcript/RNAI transcript ratios were estimated using a PhosphorImager. The gapA P1 promoter activity of the variant promoters is represented as a percentage of the gapA P1-rrnB transcript/RNAI transcript ratio of the WT promoter. The values given in (c) are mean values for three different experiments and the estimated error is indicated by vertical bars.