Bornaes C, Ignjatovic M W, Schjerling P, Kielland-Brandt M C, Holmberg S
Department of Genetics, University of Copenhagen, Denmark.
Mol Cell Biol. 1993 Dec;13(12):7604-11. doi: 10.1128/mcb.13.12.7604-7611.1993.
CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.
酿酒酵母的CHA1基因是分解代谢型L-丝氨酸（L-苏氨酸）脱水酶的基因，该酶负责丝氨酸和苏氨酸的生物降解。我们之前已经表明，CHA1基因的表达受丝氨酸和苏氨酸的转录诱导。Northern（RNA）分析表明，优质氮源的额外存在会影响诱导作用。这很可能是由于诱导物排除。为了确定顺式作用元件与CHA1启动子反式激活因子之间的相互作用，我们用CHA1启动子片段对核蛋白提取物进行了凝胶迁移实验。通过这种方法，我们鉴定出了CHA1启动子的一个蛋白质结合位点。该蛋白质的足迹包含ABF1结合位点共有序列。这种体外结合活性与CHA1是否被诱导无关。通过缺失分析，鉴定出了CHA1启动子的另外两个元件UAS1CHA和UAS2CHA，它们是CHA1基因诱导所必需的。当替换CYC1启动子的上游激活序列时，这两个序列元件中的每一个都足以赋予CYC1启动子丝氨酸和苏氨酸诱导能力。此外，在不能以丝氨酸或苏氨酸作为唯一氮源生长的cha4突变菌株中，UAS1CHA以及UAS2CHA的功能都受到阻碍。
