Zhou P B, Thiele D J
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, 48109-0606.
Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6112-6. doi: 10.1073/pnas.88.14.6112.
Metal-inducible transcription of metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements. In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1. This selection is based on the ability of the AMT1 gene product to activate expression of the C. glabrata MT-I gene in a copper-sensitive S. cerevisiae host strain. DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver to bind to both the MT-I and MT-II promoters of C. glabrata. Sequence comparison of AMT1 protein to the S. cerevisiae copper- or silver-activated DNA-binding protein, ACE1, indicates that AMT1 contains the 11 amino terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACE1. In contrast, the carboxyl-terminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACE1 known to be important for transcriptional activation. These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACE1 to sense intracellular copper levels and assume a metal-activated DNA-binding structure.
金属硫蛋白（MT）基因的金属诱导转录涉及金属反应性反式作用因子与特定启动子DNA序列元件的相互作用。在本报告中，我们展示了一种利用面包酵母酿酒酵母进行的遗传筛选，以克隆来自光滑念珠菌的一个基因，该基因编码一种名为AMT1的金属激活DNA结合蛋白。这种筛选基于AMT1基因产物在对铜敏感的酿酒酵母宿主菌株中激活光滑念珠菌MT-I基因表达的能力。使用在大肠杆菌中表达的AMT1蛋白进行的DNA结合研究表明，AMT1被铜或银激活后可与光滑念珠菌的MT-I和MT-II启动子结合。将AMT1蛋白与酿酒酵母的铜或银激活DNA结合蛋白ACE1进行序列比较，结果表明AMT1含有11个氨基末端半胱氨酸残基，已知这些残基对ACE1的金属激活DNA结合活性至关重要。相比之下，AMT1的羧基末端部分在一级结构水平上与已知对转录激活很重要的ACE1同一区域只有轻微相似性。这些结果表明，氨基末端半胱氨酸和其他保守残基在AMT1和ACE1感知细胞内铜水平并形成金属激活DNA结合结构的能力中发挥重要作用。
