CONTROL OF CITRATE SYNTHASE EXPRESSION IN YEAST

三羧酸（TCA）循环在细胞内具有核心重要性。 新陈代谢，但还没有详细的调查， 调控任何编码TCA循环酶的基因，无论是在原核生物中， 或真核细胞。 柠檬酸盐基因的调控机制 真核微生物酿酒酵母（Saccharomycescerevisiae）中的合成酶 酵母）将被定义。 柠檬酸合酶催化第一个和速率- 线粒体内TCA循环的限制步骤。 TCA循环是 非发酵性碳源的利用所需的（在 不存在葡萄糖）。 前三个TCA循环酶，柠檬酸合酶， 乌头酸酶和异柠檬酸脱氢酶也是必需的。 谷氨酸的前体α-酮戊二酸的合成。 酵母 含有两个编码功能性柠檬酸合酶的核基因：CIT 1 （主要的，线粒体同工酶）和CIT 2（次要的，非线粒体同工酶）。 两种基因的转录都被葡萄糖协同降低， 谷氨酸在生长培养基中，导致细胞经济。 以限定 这种新的调控模式所需的DNA序列，我们已经开始 构建任一基因上游的DNA缺失。 简化 为了测定基因表达，我们将CIT 1和CIT 2融合到lacZ基因上 （β-半乳糖苷酶）。杆菌 超过150个碱基的DNA序列 CIT 1和CIT 2的转录起始区上游的一对是 必要和充分（在任一方向）的基因激活， 由葡萄糖和谷氨酸调节。 进一步的缺失作图和 将使用基于多核苷酸的方法来精确定义这些DNA 序列的 反式作用因子缺陷的突变体（例如，dna结合 调节柠檬酸合酶基因所需的蛋白质）将使用 CIT 1或CIT 2 lacZ融合体和显色底物Xgal。 降解物 CIT 1（而不是CIT 2）的去抑制表达需要HAP 2，3，4 DNA- 结合复合物，它也激活各种细胞色素基因。 这是 第一个证据表明存在一种协调三氯乙酸循环调节的机制 和电子传递链，在基因表达水平上。 我们将 还可以确定是否有任何复合物在体外形成酵母蛋白质之间 和在体内调节CIT 1或CIT 2的上游DNA序列， 依赖于HAP 2、3、4基因座或由调节基因定义的其他基因座， 突变（见上文）。 我们的长期目标是了解 各种代谢和生物合成功能的要求 线粒体TCA循环的一部分被传递到核基因 编码TCA循环酶。

The tricarboxylic acid (TCA) cycle is of central importance in cellular metabolism, yet there has not been a detailed investigation of the regulation of any gene encoding a TCA cycle enzyme, in either a prokaryotic or eukaryotic cell. The mechanism of regulation of genes encoding citrate synthase in the eukaryotic microorganism Saccharomyces cerevisiae (baker's yeast) will be defined. Citrate synthase catalyzes the first and the rate- limiting step of the TCA cycle within the mitochondria. The TCA cycle is required for the utilization of nonfermentable carbon sources (in the absence of glucose). The first three TCA cycle enzymes, citrate synthase, aconitase, and isocitrate dehydrogenase, are also required for the synthesis of alpha-ketoglutarate, the precursor for glutamate. Yeast contains two nuclear genes encoding functional citrate synthase: CIT1 (major, mitochondrial isozyme) and CIT2 (minor, nonmitochondrial isozyme). Transcription of both genes is reduced synergistically by glucose and glutamate in the growth medium, resulting in cellular economy. To define DNA sequences required for this novel pattern of regulation, we have begun constructing deletions of DNA upstream of either gene. To simplify assaying gene expression we have fused CIT1 and CIT2 to the lacZ gene (beta-galactosidase) of E. coli. DNA sequences located more than 150 base pairs upstream of the transcription initiation regions of CIT1 and CIT2 are necessary and sufficient (in either orientation) for gene activation and regulation by glucose and glutamate. Further deletion mapping and oligonucleotide-based methods will be used to precisely define these DNA sequences. Mutants defective in trans-acting factors (e.g., DNA-binding proteins) required for regulation of citrate synthase genes will be using CIT1 or CIT2 lacZ fusions and the chromogenic substrate Xgal. Catabolite derepressed expression of CIT1 (but not CIT2) requires the HAP2,3,4 DNA- binding complex, which also activates various cytochrome genes. This is the first evidence for a mechanism coordinating regulation of the TCA cycle and the electron transport chain, at the level of gene expression. We will also determine whether any complexes formed in vitro between yeast proteins and upstream DNA sequences which regulate CIT1 or CIT2 in vivo are dependent on the HAP2,3,4 loci or other loci defined by regulatory mutations (see above). Our long term goal is to understand how requirements for the catabolic and biosynthetic functions of various portions of the mitochondrial TCA cycle are communicated to nuclear genes encoding TCA cycle enzymes.