REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM

脯氨酸代谢的调节和划分

基本信息

项目摘要

The long term objective of this project is to understand the molecular mechanisms that regulate gene expression in nitrogen assimilation in eukaryotic organisms. These include the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. The proline utilization enzymes are encoded by nuclear genes, translated in the cytosol, and targeted to the mitochondrion where proline is converted to glutamate. Proline, oxygen and a functional electron transport chain are essential for this process. Expression of the structural genes is regulated by an activator called PUT3. This protein is constitutively bound to the promoters of these genes, but activates their transcription only when preferred nitrogen sources are absent and proline is present. The focus of this study is to determine how the signal that proline is available for utilization is transmitted through the PUT3 protein to the transcriptional machinery. The functional domains of the activator protein will be defined by an analysis of mutant PUT3 genes that encode activator-defective proteins, construction of site-directed mutations in specific domains of the gene, exchange of domains with those of GAL4, a well-characterized activator, and measurement of the effects of these alterations on DNA binding, transcriptional activation, proline-responsiveness and dimerization of the protein. The PUT3 protein will be purified using DNA-affinity chromatography and examined for post-translational modifications (phosphorylation, glycosylation, proline-binding and association with other proteins) using standard biochemical methods. A new class of mutations in negative regulators controlling this pathway has been identified and will be characterized by genetic and molecular methods. One member of this class, the product of the PUT6 gene, apparently works through PUT3 in controlling proline utilization, but is not specific to this pathway. Genes that interact with PUT3 will be identified by the isolation of high copy and extragenic suppressors of put3 and PUT3c mutations. This system provides a useful model to understand how changes in the cellular environment are signalled to the elements of a metabolic pathway in eukaryotic organisms.
该项目的长期目标是了解分子 调节氮同化基因表达的机制 真核生物 这些包括调节蛋白的作用, 酶和底物的区室化,以及代谢物在 代谢途径的整体功能。 脯氨酸利用率 酵母酿酒酵母(Saccharomycescerevisiae)作为本研究的模型系统。 脯氨酸利用酶由核基因编码,翻译 在胞质溶胶中,并靶向脯氨酸所在的线粒体, 转化为谷氨酸 脯氨酸、氧和一个功能性电子 运输链对这一过程至关重要。 表达 结构基因是由一种叫做PUT3的激活剂调控的。 这种蛋白质 与这些基因的启动子组成型结合,但激活 它们的转录仅在优选的氮源不存在时进行, 脯氨酸存在。 这项研究的重点是确定如何 脯氨酸可用于利用的信号通过 PUT3蛋白的转录机制。 功能 激活蛋白的结构域将通过突变体的分析来定义。 编码激活因子缺陷蛋白的PUT3基因, 基因特定区域的定点突变, 结构域与GAL4的结构域相同,GAL4是一种充分表征的激活剂, 测量这些改变对DNA结合的影响, 转录激活,脯氨酸反应和二聚化 蛋白质。 将使用DNA亲和层析纯化PUT3蛋白。 层析并检查翻译后修饰 (磷酸化、糖基化、脯氨酸结合和与 其它蛋白质)。 一类新的 控制这一途径的负调节因子的突变已经被 将通过遗传和分子方法进行鉴定和表征。 这类基因中的一个成员,PUT6基因的产物,显然起作用 通过PUT 3控制脯氨酸利用,但并不特异于 这条路。 与PUT3相互作用的基因将通过免疫组织化学鉴定。 Put 3和Put 3c高拷贝和基因外抑制子分离 突变。 这个系统提供了一个有用的模型来理解如何改变 在细胞环境中的信号传递给代谢的元素, 在真核生物中的途径。

项目成果

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MARJORIE C BRANDRISS其他文献

MARJORIE C BRANDRISS的其他文献

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{{ truncateString('MARJORIE C BRANDRISS', 18)}}的其他基金

REGULATION & COMPARTMENTATION IN PROLINE METABOLISM
规定
  • 批准号:
    3298637
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION & COMPARTMENTATION IN PROLINE METABOLISM
规定
  • 批准号:
    3298634
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM
脯氨酸代谢的调节和划分
  • 批准号:
    2180570
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM
脯氨酸代谢的调节和划分
  • 批准号:
    2900701
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM
脯氨酸代谢的调节和划分
  • 批准号:
    2180572
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM
脯氨酸代谢的调节和划分
  • 批准号:
    6180105
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION & COMPARTMENTATION IN PROLINE METABOLISM
规定
  • 批准号:
    3298632
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION & COMPARTMENTATION IN PROLINE METABOLISM
规定
  • 批准号:
    3298636
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM
脯氨酸代谢的调节和划分
  • 批准号:
    3298633
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:
REGULATION & COMPARTMENTATION IN PROLINE METABOLISM
规定
  • 批准号:
    3298635
  • 财政年份:
    1988
  • 资助金额:
    $ 27.5万
  • 项目类别:

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DNA footprinting of a plant defense gene family; to support visit by A.M. Yorkin, Department of Genetics, St. Petersburg State University, St. Petersburg, Russia
植物防御基因家族的 DNA 足迹;
  • 批准号:
    147394-1992
  • 财政年份:
    1993
  • 资助金额:
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  • 项目类别:
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