Mellor J, Jiang W, Funk M, Rathjen J, Barnes C A, Hinz T, Hegemann J H, Philippsen P
Department of Biochemistry, Oxford, UK.
EMBO J. 1990 Dec;9(12):4017-26. doi: 10.1002/j.1460-2075.1990.tb07623.x.
Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met- phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI-carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy-terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix-loop-helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy-terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino-terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.
酿酒酵母的着丝粒和几个启动子含有一个高度保守的八核苷酸RTCACRTG，称为CDEI。通过生化、遗传和结构分析，我们发现同一种蛋白质在体内与着丝粒和启动子中的CDEI位点结合。这种蛋白质，称为着丝粒启动子因子CPF1，以二聚体形式结合DNA。该基因的失活并不致命，但会导致着丝粒功能部分丧失并导致甲硫氨酸缺陷型表型。由于CPF1失活导致的染色质结构变化在着丝粒和几个携带CDEI的启动子（如MET25、TRP1、GAL2）处可见。然而，启动子活性受到多种方式的影响，目前很难描述CPF1在基因表达中的功能。克隆基因的序列在羧基末端部分揭示了两个潜在的两亲性螺旋，前面是一段带正电荷的氨基酸序列，与最近在高等真核生物中控制组织特异性转录的因子中鉴定出的螺旋-环-螺旋结构域非常相似。缺乏该结构域的CPF1羧基末端截短体不再与CDEI结合。CPF1的氨基末端一半带有两簇带负电荷的氨基酸残基。令人惊讶的是，这些簇的缺失仍然使细胞为甲硫氨酸野生型，并且仅导致着丝粒活性略有下降。
