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首页> 外文学位 >Highly-efficient and specific mRNA cleavage by a non-canonical endoribonuclease: A kinetic investigation of the ribosome-dependent toxin RelE.
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Highly-efficient and specific mRNA cleavage by a non-canonical endoribonuclease: A kinetic investigation of the ribosome-dependent toxin RelE.

机译：非规范性内切核糖核酸酶的高效和特异性mRNA切割：核糖体依赖性毒素RelE的动力学研究。

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摘要

RelE and related prokaryotic toxins have been implicated in the regulation of diverse cellular processes, including persistence. During nutrient stress, RelE cleaves A-site mRNA, leading to a global inhibition of translation. Highly conserved RelE residues- Lys52, Lys54, Arg61, Arg81, and Tyr87- surround the mRNA scissile phosphate and ribosomal contacts further contribute to substrate positioning, but the active-site composition lacks obvious candidates for general acid-base catalysis. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active-site. RelE rapidly and selectively cleaves ribosomal A-site mRNA at a rate similar to traditional ribonucleases. The mutant rate effects for residues Arg61 and Arg81 are consistent with the catalytic roles proposed for each arginine in charge stabilization and leaving group protonation. Mutation of either lysine decreased catalysis 103-fold. On the basis of structural proximity, Lys52 may be responsible for proton abstraction at the 2'-hydroxyl, with Lys54 contributing to phosphate charge stabilization. In the absence of a robust general base, RelE may principally promote catalysis via transition state charge stabilization and leaving group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of a novel endoribonuclease involved in prokaryotic regulation.
机译：RelE和相关的原核毒素与多种细胞过程的调控有关，包括持久性。在营养胁迫期间，RelE切割A位mRNA，从而导致翻译的整体抑制。高度保守的RelE残基-Lys52，Lys54，Arg61，Arg81和Tyr87-围绕mRNA易裂的磷酸酯酶和核糖体接触进一步促进了底物的定位，但是活性位点的组成缺乏一般酸碱催化作用的明显候选物。我们使用单周转动力学测定来评估RelE活性位点中单个残基的催化重要性。 RelE以类似于传统核糖核酸酶的速率快速，选择性地切割核糖体A位mRNA。残基Arg61和Arg81的突变率效应与每个精氨酸在电荷稳定和离去基团质子化中提出的催化作用一致。任一赖氨酸的突变均可将催化作用降低103倍。基于结构上的接近，Lys52可能负责2'-羟基处的质子提取，而Lys54有助于稳定磷酸盐电荷。在缺乏稳固的通用碱的情况下，RelE可以主要通过过渡态电荷稳定化和离开基团质子化促进催化作用，此外还可以与核糖体协同实现在线底物定位。该动力学分析补充了结构信息，为理解参与原核生物调节的新型核糖核酸内切酶的分子机制提供了基础。

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  • 作者

    Griffin, Meghan Ann.;

  • 作者单位

    Yale University.;

  • 授予单位 Yale University.;
  • 学科 Biochemistry.;Biophysics.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 184 p.
  • 总页数 184
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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