Daniels Patrick J, Bittel Doug, Smirnova Irina V, Winge Dennis R, Andrews Glen K
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7421, USA.
Nucleic Acids Res. 2002 Jul 15;30(14):3130-40. doi: 10.1093/nar/gkf432.
The zinc finger protein, metal response element-binding transcription factor-1 (MTF-1) regulates the expression of genes in response to metal ions and oxidative stress. The precise mechanisms by which this occurs are not understood. To further examine this problem, mouse MTF-1 was expressed in Saccharomyces cerevisiae and tested for the ability to activate metal response element-driven reporter gene expression. Zinc was an effective inducer of reporter gene expression. In general, the magnitude of zinc induction was dependent on the concentration of zinc in the culture medium, but independent of the amount of MTF-1 expression. Zinc induction also occurred with either integrated or episomal reporter plasmids containing the native mouse metallothionein-I proximal promoter. Deletion of fingers 5 and 6 of MTF-1, which function in a zinc-dependent manner to stabilize the DNA-binding activity of the protein in vitro, did not diminish the zinc induction of either episomal or integrated promoters. However, a Gal4 DNA-binding domain- MTF-1 fusion protein, which binds constitutively to the Gal4-responsive promoter, was not zinc inducible but caused constitutive activation of reporter gene expression. This suggests that zinc activation of the DNA-binding activity of MTF-1 is the rate limiting step in its metalloregulatory function in yeast. In contrast, MTF-1 was not responsive to either cadmium or hydrogen peroxide, suggesting that distinct co-activators or signal transduction cascades not found in yeast are required to mediate MTF-1 activation of gene expression by this toxic metal and by oxidative stress.
锌指蛋白金属反应元件结合转录因子1(MTF-1)可响应金属离子和氧化应激调节基因表达。其发生的精确机制尚不清楚。为了进一步研究这个问题,在酿酒酵母中表达了小鼠MTF-1,并测试其激活金属反应元件驱动的报告基因表达的能力。锌是报告基因表达的有效诱导剂。一般来说,锌诱导的程度取决于培养基中锌的浓度,但与MTF-1的表达量无关。含有天然小鼠金属硫蛋白-I近端启动子的整合型或附加型报告质粒也能发生锌诱导。MTF-1的第5和第6个锌指在体外以锌依赖的方式发挥作用以稳定蛋白质的DNA结合活性,缺失这两个锌指并不会减弱附加型或整合型启动子的锌诱导。然而,一种组成性结合Gal4反应性启动子的Gal4 DNA结合结构域-MTF-1融合蛋白不能被锌诱导,但会导致报告基因表达的组成性激活。这表明MTF-1的DNA结合活性的锌激活是其在酵母中的金属调节功能的限速步骤。相比之下,MTF-1对镉或过氧化氢均无反应,这表明酵母中不存在的独特共激活因子或信号转导级联反应是介导MTF-1被这种有毒金属和氧化应激激活基因表达所必需的。