Fig. 3.

Loz1 is required for zinc-dependent transcriptional regulation of gene expression. (A) Wild-type and loz1Δ cells containing the empty vector, adh4-lacZ, or SPBC1348.06c-lacZ reporters were grown in ZL-EMM supplemented with 0 μM (-Zn) or 200 μM zinc (+Zn) before cells were harvested for β-galactosidase assays. (B) Wild-type and loz1Δ cells containing the indicated reporter constructs were grown as described in A. Cells were harvested and β-galactosidase activity was measured by standard procedures. (C) SDS/PAGE analysis of crude lysates from isopropyl β-D-1-thiogalactopyranoside-induced E. coli cells containing the Pet32a empty vector (TH tag), Pet32a-Loz1 (TH-Loz1), or Pet32a-Loz1Cys470Gly (TH-Loz1Cys470Gly) (lanes 2–4), or the Ni-NTA affinity-purified TH tag, TH-Loz1, or TH-Loz1Cys470Gly (lanes 5–7). Proteins were visualized by using Coomassie blue. The sizes of a molecular mass ladder (lane 1) are shown in kilodaltons on the left. (D) Sequence of the probes used for EMSA. All probes used were double-stranded; the sequence of the 5′ strand for each probe is shown. The GN(A/C)GATC element is boxed. Mutations in probe 5mut are highlighted in bold. (E) A representative EMSA using 0.1 ng of P32-labeled double-stranded probes T8 and T5 in the presence of increasing levels (0, 0.5, 1, 2.5, and 5 μg) of poly dIdC. (F) A representative EMSA using 0.1 ng of P32-labeled double-stranded probes T5, T5mut, and T8, and affinity-purified TH tag, TH-tagged Loz1, and TH-Loz1Cys470Gly in the presence of 1 μg of poly dIdC. Free probe and bound probe are indicated.