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Recognition of the RNA polymerase II CTD by transcription termination factors.

机译：通过转录终止因子识别RNA聚合酶II CTD。

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摘要

The phosphorylation state of the C-terminal domain (CTD) of RNA polymerase II controls the assembly of RNA processing and transcription factors on the elongating enzyme. I explored the molecular basis for the recognition of specific CTD phosphoisoforms by CTD-interacting domains by probing the transcription termination and 3'-end processing factors Rtt103 and Pcf11 with an exhaustive panel of CTD mimics containing all possible phosphorylation states. When presented with a single binding site composed of two heptad repeats, Rtt103 binds to Ser2 phosphorylated (Ser2P) peptide strongly and specifically, while Pcf11 does so much less well. A single amino acid change reverses the specificity of these two proteins in vitro and in vivo. However, both Pcf11 and Rtt103 cooperatively recognize peptides containing two or more binding sites. My data suggests that the cooperativity observed for Pcf11 is critical for its proper recruitment to bona-fide transcription termination sites, while preventing transcription at cryptic sites. My work demonstrates for the first time that neighboring CTD repeats can function together to create a precisely tuned switch that can achieve a rapid, sigmoidal response allowing for precise control of co-transcriptional RNA processing.;The phosphorylation state of the CTD is dynamically altered during transcription by a number of CTD kinases and phosphatases. I have performed a number of initial experiments on the CTD Ser5P phosphatase, Ssu72, in order to obtain a suitable amount of protein for structure determination. Using a combination of sequence alignment and structure prediction, I was able to produce a soluble yeast Ssu72 construct. Also, I was able to produce yeast Ssu72 co-expressed with its binding partner Pta1. In addition to my work on yeast Ssu72, I also have completed partial assignments of the human Ssu72 protein.
机译：RNA聚合酶II的C末端结构域（CTD）的磷酸化状态控制着延伸酶上RNA加工和转录因子的装配。我通过用详尽的包含所有可能的磷酸化状态的CTD模拟物探测转录终止和3'-末端加工因子Rtt103和Pcf11，探索了CTD相互作用域识别特定CTD磷酸同工型的分子基础。当出现由两个七肽重复序列组成的单个结合位点时，Rtt103强烈且特异性地结合到Ser2磷酸化（Ser2P）肽上，而Pcf11的结合则差很多。单个氨基酸变化会在体外和体内逆转这两种蛋白质的特异性。但是，Pcf11和Rtt103都可以协同识别含有两个或多个结合位点的肽。我的数据表明，观察到的Pcf11的协同作用对于正确募集至真正的转录终止位点至关重要，同时又可以防止在隐蔽位点进行转录。我的工作首次证明相邻的CTD重复序列可以共同发挥作用，从而创建一个精确调节的开关，从而实现快速的S型响应，从而可以精确控制共转录RNA的加工过程;在此过程中，CTD的磷酸化状态会动态改变。许多CTD激酶和磷酸酶的转录。我已经对CTD Ser5P磷酸酶Ssu72进行了许多初步实验，以便获得适量的蛋白质用于结构确定。使用序列比对和结构预测的组合，我能够生产可溶性酵母Ssu72构建体。而且，我能够生产与其结合伴侣Pta1共表达的酵母Ssu72。除了研究酵母Ssu72之外，我还完成了人Ssu72蛋白的部分分配。

著录项

  • 作者

    Lunde, Bradley M.;

  • 作者单位

    University of Washington.;

  • 授予单位 University of Washington.;
  • 学科 Chemistry Biochemistry.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 165 p.
  • 总页数 165
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:19

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