Anderson S F, Steber C M, Esposito R E, Coleman J E
Department of Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Protein Sci. 1995 Sep;4(9):1832-43. doi: 10.1002/pro.5560040918.
UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes. UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors. This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster. In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein. At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149). The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone. DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence.
UME6是一种来自酿酒酵母的由836个氨基酸组成的蛋白质，它作为几个早期减数分裂基因的阻遏物和激活物发挥作用。UME6在靠近C端的位置含有氨基酸序列-771C-X2-C-X6-C-X6-C-X2-C-X6-C-，其中六个半胱氨酸残基的间距与真菌转录因子的39个N端富含半胱氨酸的DNA结合亚结构域中的间距相同。该序列已在GAL4和其他蛋白质中被证明可形成锌双核簇。尽管位置不同，但在UME6的最后111个氨基酸残基内克隆并过量表达的富含C的序列UME6(111)形成了一个双核簇，并表现出对URS1 DNA序列的锌依赖性结合。后者TAGCCGCCGA是UME6对减数分裂特异性基因进行阻遏或激活所必需的。UME6(111)含有1.8±0.4摩尔锌/摩尔蛋白质，锌可以被镉交换，产生一种含有1.9±0.1摩尔镉/摩尔蛋白质的蛋白质。在5℃下，113Cd2UME6(111)显示出两个113Cd NMR信号，化学位移分别为699和689 ppm，与113Cd2GAL4(149)观察到的信号相似。这些化学位移的大小表明每个113Cd核与四个-S-配体配位，这与一个113Cd2簇结构兼容，其中两个硫醇盐来自桥连配体。整个UME6基因已被克隆并过量表达，并且它与URS1序列的结合比单独的锌双核簇结构域更紧密。UME6在含URS1的DNA上的DNase I足迹表明，该蛋白质保护URS1序列内5'-CCGCCG-3'区域的磷酸二酯键。
