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Rapid Steps in the glmS Ribozyme Catalytic Pathway: Cation and Ligand Requirements

机译：GLMS核酶催化途径中的快速步骤：阳离子和配体要求

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摘要

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg2+-containing buffers effectively isolates the rapid ligand binding and catalytic events (k(obs) > 60 min(-1)) from rate-limiting folding (k(obs) < 4 min(-1)). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure but are unable to promote ligand binding and catalysis rates of > 4 min(-1). Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand-binding pocket. In further support of this hypothesis, our data show that a nonphosphorylated analogue of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity, but the rapid initiation of catalysis upon the addition of ligand is stricter.
机译：GLMS核酶是一种在众多革兰氏阳性细菌中发现的保守核糖，并响应葡糖胺6-磷酸的细胞浓度（GLCN6P）。 GLCN6P结合促进GLM mRNA的5'UTR中的特异性自切割，导致基因表达的下调。当测量切割反应途径的速率限制折叠步骤时，先前已经显示出胶质核酶缺乏强的阳离子特异性。这不提供关于快速配体结合化学催化事件期间GLMS核酶的阳离子和配体特异性的数据。在Mg 2 +临床缓冲液中将核酶的预热有效地将快速配体结合和催化事件（K（OB）> 60 min（-1））从速率限制折叠（K（OBS）<4分钟（-1））分离。在这里，我们采用这种实验设计来测定快速配体结合和催化的阳离子和配体要求。我们表明，单价阳离子的摩尔浓度也能够诱导天然GLCN6P结合结构的形成，但不能促进> 4分钟（-1）的配体结合和催化速率。我们的数据表明，具有晶体阳离子的二价阳离子的唯一强制作用是在配体结合口袋中的GLCN6P的磷酸盐部分配制。在对该假设的进一步支持中，我们的数据表明，GLCN6P，葡糖胺的非磷酸化类似物不能在二价阳离子存在下促进快速配体结合和催化。因此，核酶的折叠是相对独立于阳离子同一性，但在加入配体时催化的快速启动是更严格的。

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  • 来源
    《Biochemistry》 |2011年第13期|共10页
  • 作者

    Brooks KM; Hampel KJ;

  • 作者单位

    Department of Microbiology and Molecular Genetics The Markey Center for Molecular Genetics Stafford Hall 95 Carrigan Drive University of Vermont Burlington Vermont 05401 United States;

    Department of Microbiology and Molecular Genetics The Markey Center for Molecular Genetics Stafford Hall 95 Carrigan Drive University of Vermont Burlington Vermont 05401 United States;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    glmS Ribozyme Catalytic Pathway; Cation; Ligand Requirements;

    机译：GLM核酶催化途径;阳离子;配体要求;

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