FIG. 3.

Identification of the YusO binding region. (A) Determination of the transcription start site of the yusOP operon. Total RNAs (45 μg) of strains 168 (wild type, lane 1) and YUSOd (yusO::pMUTIN2, lane 2) were reverse transcribed to generate runoff cDNA (bold arrow). Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the same primer used for reverse transcription. The partial nucleotide sequence of the coding strand corresponding to the ladders is shown, where the −10 regions are underlined and the transcription start sites (+1) are boxed. (B) DNase I footprinting of YusO in the yusOP promoter region. DNA probes corresponding to the coding or noncoding strand of the yusOP promoter region were 5′ end labeled, and each was incubated at a final concentration of 0.8 nM with a crude extract from E. coli BL21(DE3) expressing yusO in the absence (lanes 1 and 5) or presence (lane 2, 2.6 μM; lane 3, 1.3 μM; lane 4, 0.65 μM as a dimer) of crude YusO protein. After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the corresponding 5′-labeled primer. Nucleotide sequences protected by YusO are indicated on the right of each panel. (C) Organization of the yusOP promoter region. The stop codon of the yusN gene and the −35 and −10 regions of the yusOP promoter are underlined. The transcription start site (+1) of the yusOP operon is shown by capital boldface letter. The Shine-Dalgarno (SD) sequence of yusO is boxed, and the three inverted repeat sequences, IR1, IR2, and IR3, are indicated by pairs of facing arrows with bold letters showing the matching bases. The open reading frames of yusN and yusO genes are depicted by thick lines. The protected regions in the coding and noncoding strands are indicated by gray bars.