Figure 5: Zinc-dependent formation of multimeric Zur-zitB DNA complexes in vitro and the contribution of Zur-box upstream region on zitB activation in vivo.

(a) EMSA analysis of Zur binding on 33 bp zitB DNA probe in comparison with the complex on 25 bp zitB DNA. Increasing amounts of zinc (0, 0.1, 0.5, 1.0, 2.5, 5, 10 and 20 μM) were included in the binding buffer with 90 nM Zur. The molecular weights of the retarded bands were estimated from electrophoretic mobility on native PAGE with different acrylamide percentages (Supplementary Fig. 9), and were marked as T (tetramer) or D (dimer). (b) EMSA analysis on the 114 bp zitB probe (from −148 to −35 nt). Increasing amounts of zinc (0, 0.5, 1, 5, 10 and 20 μM) were included in the binding buffer with 90 nM Zur. Based on the estimated molecular weights from native PAGE mobility, the retarded bands were indicated by O for octamer, and D for dimer. (c) Expression of GUS reporter gene linked with the zitB promoter region from +50 to −60 nt (pzitB-60GUS) or to −228 nt (pzitB-228GUS). S. coelicolor cells containing the chromosomally integrated reporter gene were either non-treated or treated with 10 μM TPEN or 100 μM ZnSO₄ for 30 min. Quantitation of S1 mapping results were done from three independent experiments, and the relative expression values were presented by taking the non-treated level as 1.0. The P values of all the relative measurements except zinc-treated pzitB-60GUS were ≤0.001 by Student’s t-test. (d) In vitro transcription assays of the zitB and znuA promoters in the presence of purified Zur (50 nM) and RNA polymerase core enzyme (E. coli) and the housekeeping sigma factor HrdB (S. coelicolor). Varying amounts of ZnSO₄ (0, 1, 5, 10, 15 and 20 μM) were added in the transcription buffer. Predicted lengths of the zitB and znuA transcripts are 52 nt (left arrow) and 87 nt (right arrow), respectively.