Table 2 Promoter activity determined by LacZ reporter fusion analysis LacZ fusion Plasmid copy number (WT/Δzur) Normalized Miller Units Fold change (Δzur/WT) WT-znuC 5.45 ± 0.73 6343.95 ± 237.68 2.60 Δzur-znuC
16507.10 ± 344.19 WT-znuA 11.52 ± 0.92 12281.64 ± 428.30 7.77 Δzur-znuA 95498.09 ± 1962.30 WT-ykgM 3.09 ± 0.88 118.64 ± 6.77 4.71 Δzur-ykgM 559.29 ± 28.14 Notes: The promoter DNA regions upstream znuC, znuA and ykgM were cloned into the pRS551 plasmid, respectively, to fuse with the promoterless lacZ gene. β-Galactosidase activity (miller units) was detected to represent the promoter activity. Copy number of recombinant pRS551 was determined by real-time quantitative PCR, with the primers specific for selleck the borne lacA gene. The detecting Fer-1 fold change of plasmid copy number was set to be 1 to generate
a normalization factor that was subsequently used for generating the normalized fold change of promoter activity (miller units) in WT in relative to Δzur. Structural organization of Zur-dependent znuCB, znuA and ykgM-rpmJ2 promoters Primer extension assay was performed to determine the transcription start sites of znuC, znuA and ykgM (Fig. 4). A strong primer extension product was detected for both znuC and ykgM, while three primer extension products were detected for znuA. Since the shorter extension products might represent the premature stops due to difficulties of polymerase in passing difficult sequences, only the longest product was chosen for the transcription start site of znuA. Accordingly, a transcription start site was identified for each of the three genes, and thereby a Zur-dependent promoter was transcribed for each of them. The nucleotide number of each transcription start site was taken as ‘+1′, and the -10 and -35 core promoter elements recognized by sigma factor 70 were predicted upstream the transcription start sites. Figure 4 Primer extension assays. Primer extension assays were performed for znuC, znuA and ykgM, by using Interleukin-3 receptor RNA isolated from the exponential-phase of both WT and Δzur grown in TMH ATR inhibitor medium with 5 mM of Zn2+. An oligonucleotide
primer complementary to the RNA transcript of each gene was designed from a suitable position. The primer extension products were analyzed with 6% acrylamide sequencing gel. Lanes C, T, A and G represented the Sanger sequencing reactions. On the right side, DNA sequences were shown from the bottom (5′) to the top (3′), and the transcription start sites were underlined. To precisely determine the Zur binding sites of znuCB, znuA and ykgM-rpmJ2, DNase I footprinting assay was performed in the presence of zinc (both coding and noncoding strands) (Fig. 5). DNase I footprinting results confirmed the binding of Zur to these promoter regions in vitro. Zur protected a distinct DNA region (i.e. Zur binding site) against DNase I digestion in a dose-dependent pattern for ykgM (Fig. 5).