CONTROL OF CITRATE SYNTHASE EXPRESSION IN YEAST

三羧酸(TCA)循环在细胞内起着重要的作用 新陈代谢，但还没有详细的调查 对编码TCA循环酶的任何基因的调节，在原核生物中 或者真核细胞。编码柠檬酸的基因的调控机制 真核微生物酿酒酵母(Baker‘s)中的合成酶 酵母)将被定义。柠檬酸合成酶催化第一个和速率- 线粒体内TCA循环的限制步骤。三氯乙烷周期为 利用非发酵碳源所需的碳源(在 缺乏葡萄糖)。前三种三氯乙酸循环酶，柠檬酸合成酶， 乌头酸酶和异柠檬酸脱氢酶也是必需的 谷氨酸前体α-酮戊二酸的合成。酵母菌 含有两个编码功能性柠檬酸合成酶的核基因：CIT1 CIT2(次要，非线粒体同工酶)。 这两个基因的转录被葡萄糖和 在生长介质中含有谷氨酸，导致细胞经济。要定义 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。简单化 在基因表达分析中，我们将CIT1和CIT2与LacZ基因融合 (β-半乳糖苷酶)。位于150个碱基以上的DNA序列 CIT1和CIT2转录起始区上游的对是 基因激活的必要条件和充分条件(在任何方向上) 受葡萄糖和谷氨酸的调节。进一步的删除映射和 基于寡核苷酸的方法将被用来精确地定义这些DNA 序列。反式作用因子缺陷的突变体(例如，DNA结合 调节柠檬酸合成酶基因所需的蛋白质)将使用 CIT1或CIT2 LacZ融合及显色底物XGal。分解代谢 CIT1(而不是CIT2)的去抑制表达需要HAP2，3，4 DNA- 结合复合体，它还激活各种细胞色素基因。这是 三氯乙烷循环协调机制的第一个证据 以及电子传输链，在基因表达水平上。我们会 也要确定在体外酵母蛋白之间是否形成了任何复合体 在体内调节CIT1或CIT2的上游DNA序列是 依赖于HAP2、3、4基因座或由调节性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 对各种生物合成和分解代谢功能的要求 线粒体TCA循环的一部分与核基因相连 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.