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. 2010 Oct 7;6(10):e1001149. doi: 10.1371/journal.pgen.1001149

Figure 2. Primer extension analysis and mutagenesis strategy of the intS promoter.

Figure 2

A. The labeled primer was annealed to RNA extracted from MC4100 (lane 1), LCB1024 (ΔintS, lane 2) and LCB1019 (ΔKplE1 prophage) harboring pattL-gfp plasmid (lane 3) strains grown aerobically and extended with reverse transcriptase. Lanes G, T, C and A are a sequencing ladder of the attL DNA region. A complementary sequence is indicated between dotted lines. B. Schematic representation of IntS and TorI binding sites on attL that overlap the intS promoter region (I, TorI; P', IntS arm-type and O, IntS core-type). The −35 and −10 boxes and ATG of intS are indicated. C. intS promoter sequence. This sequence corresponds to the attL region cloned as a reference for PintS promoter studies (pattL-gfp, positions −223 to +64 relative to the ATG). The bold letters show the putative −35 and −10 boxes and ATG (2464.565 kb on the MG1655 E. coli chromosome) of the intS gene. Protein binding sites' sequences are indicated (red boxes, TorI; blue arrows, IntS arm-type; and brackets, IntS core-type) as well as the two initiation transcription sites (V). The mutagenesis of the protein binding sites was performed by overlap extension PCR to generate mutations (*). Substitutions are indicated between dotted lines. Contrary to the P'1* mutation that affects the consensus of IntS arm-type and the −10 box, P'1-(A, B or C) constructions do not affect the −10 box. The 3′ end of the argW tRNA gene sequence is underlined (13 nucleotides of argW are missing at the 5′ end).