Figure 1
Identification of 5′ and 3′ ends of RyeA and SdsR. (A) Schematic representation of the region containing the sdsR/ryeA locus and its nucleotide sequence. Promoter regions (−35 and −10) of sdsR and ryeA are underlined, and their transcription start sites are indicated by arrows. (B) Primer extension analysis of ryeA-CAT and sdsR-CAT fusion transcripts. The regions +341 to +132 (containing the ryeA promoter) and −79 to +11 (containing the sdsR promoter) were cloned into pKK232-8 to generate ryeA-CAT and sdsR-CAT plasmids, respectively. Total cellular RNA extracts were prepared from either MG1655 cells containing the ryeA-CAT or sdsR-CAT plasmid grown for 2 h, 6 h, or 10 h at 37 °C. The 32P-labeled primer CAT_R was used to analyze ryeA-CAT and sdsR-CAT fusion transcripts. Primer extension products were analyzed on a 5% polyacrylamide sequencing gel containing 8 M urea. The DNA ladders (G, A, T and C) were prepared by dideoxy sequencing using the template plasmid DNA and the same primer. Loading amounts are indicated below the lanes. The transcription start nucleotides are indicated by arrows. (C) RACE analysis. The 5′ or 3′ RACE products (primary PCR products or nested PCR products) were analyzed on 2% agarose gels. Predicted RACE products were indicated by a, b, c, and d. For 5′ RACE, E. coli RNA pyrophosphatase (RppH)-treated RNA and untreated RNA were used. M, 100 bp size markers; R, RyeA; S, SdsR.
