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首页> 外文学位 >Identification of factors regulating transcription termination by RNA polymerase II in Saccharomyces cerevisiae.
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Identification of factors regulating transcription termination by RNA polymerase II in Saccharomyces cerevisiae.

机译：鉴定酿酒酵母中RNA聚合酶II调控转录终止的因素。

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摘要

Efficient termination of transcription plays an important role in cell homeostasis by preventing spurious transcription that might interfere with cis-acting regulatory elements found downstream of genes, by ensuring synthesis of functional transcripts, and by promoting recycling of factors needed for a new transcription cycle.;Another technique was high-copy suppression of growth and termination defects of mutants encoding factors required for snoRNA termination leading to the identification of Sen1, which, by re-anchoring the yeast type I protein phosphatase to the machinery required for snoRNA termination, restored a dephosphorylation-dependent event regulating this process.;Finally, systematic screening of mutants for termination defects of a reporter construct also identified new factors required for termination of mRNAs, including Sen1. Genetic and biochemical techniques were used to support a general role in mRNA termination for Sen1 and proved that termination of different classes of transcripts synthesized by RNAPII occurs through a common mechanism.;I used both biochemical and genetic approaches to identify new factors required for termination by RNAPII. One was isolation by tandem affinity purification of complexes containing known and putative termination factors and identification by mass spectrometry of the specifically associated proteins. Systematic purification of these associated proteins allowed identification of the core components of protein complexes, as well as less stable bridges between complexes. In addition, purification of complexes from strains in which a component of the complex was absent or mutated permitted identification of factors playing structural roles within the complex, as well as interactions between the components of the complex.
机译：有效终止转录在细胞稳态中起着重要作用，它可以防止可能干扰基因下游顺式作用调控元件的伪转录，通过确保功能性转录本的合成以及促进新转录周期所需因子的循环利用来进行。另一种技术是高拷贝抑制snoRNA终止所需的编码因子的突变体的生长和终止缺陷的复制，从而导致Sen1的鉴定，Sen1通过将酵母I型蛋白磷酸酶重新锚定到snoRNA终止所需的机制而恢复了去磷酸化依赖的事件调节这个过程。最后，系统筛选突变体的报告基因构建体的终止缺陷还确定了终止Sens所需的新因子，包括Sen1。遗传和生物化学技术被用来支持Sen1在mRNA终止中的一般作用，并证明RNAPII合成的不同类别的转录物的终止是通过共同的机制发生的。 RNAPII。一种是通过串联亲和纯化含有已知和推定终止因子的复合物进行分离，然后通过质谱法鉴定特异性结合的蛋白质。这些相关蛋白质的系统纯化可以鉴定蛋白质复合物的核心成分，以及复合物之间不太稳定的桥。另外，从其中不存在复合物组分或突变的菌株中纯化复合物允许鉴定在复合物内发挥结构作用的因子以及复合物的组分之间的相互作用。

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  • 作者

    Nedea, Eduard.;

  • 作者单位

    University of Toronto (Canada).;

  • 授予单位 University of Toronto (Canada).;
  • 学科 Biology Molecular.;Biology Microbiology.;Biology Genetics.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 236 p.
  • 总页数 236
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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