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首页> 外文学位 >Origins of translation machinery: Aminoacylation of minimalist tRNA(leu) molecules and non-ribosomal peptide bond formation.
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Origins of translation machinery: Aminoacylation of minimalist tRNA(leu) molecules and non-ribosomal peptide bond formation.

机译：翻译机制的起源：极简tRNA（leu）分子的氨基酰化作用和非核糖体肽键的形成。

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摘要

A series of RNA analogs of Escherichia coli tRNA leu with specific domain deletions and/or point mutations were synthesized. These RNAs were used to study the recognition requirements for aminoacylation by leucyl-tRNA synthetase (LeuRS). The anticodon stem-loop and variable arm domains were both found to be separately dispensable for Ieucylation. Simultaneous deletion of both domains further reduced aminoacylation. It was possible to partially restore activity by using alternative sequences in the nucleotide linker region between the remaining D-stem and T-stem. Point mutations in the D- and T-loops in one of the analogs completely abolished leucylation. Changing A73 to C73 in this minimalist variant also abolished leucylation, confirming that the discriminator base was a major identity element. These results suggest that tertiary interactions in the core region of tRNA leu are essential for recognition by LeuRS. Hydrolytic editing assays with a LeuRS mutant enzyme and each of the aminoacylatable tRNAleu analogs suggest that the variable loop interacts with the enzyme's editing site.;Several avenues by which aminoacylated RNAs might participate in peptide bond formation in the absence of ribosomal components were also investigated. The potential for 5'-leucyl-adenylate anhydrides to form dipeptides was tested. Leu-Leu dipeptides and leucine polypeptides were detected by thin layer chromatography. A published claim that dipeptides could be formed in the presence of aminoacylated tRNA, a corresponding mRNA template, Mg 2+, and Ala-His dipeptide acting as a catalyst was examined in detail. The reactions published were duplicated using [14C]-leucine-tRNA leu and poly-AUUU mRNA, as well as alternative catalysts or substrates. Leu-Leu dipeptide synthesis did not occur in the presence of Ala-His, although two unidentified by-products were formed during the reaction. Unknown product 1 was shown to be a leucyl-ethyl-ester formed by ethanolysis. Unknown product 2 was identified as adenosine-leucine. These results strongly suggest that the published claims may be artifactual.
机译：合成了具有特定结构域缺失和/或点突变的一系列大肠杆菌tRNA leu RNA类似物。这些RNA用于研究亮氨酰tRNA合成酶（LeuRS）对氨基酰化的识别要求。发现反密码子茎环和可变臂结构域对于Ieucylation都是分别可有可无的。两个结构域的同时缺失进一步减少了氨基酰化。通过使用其余D-茎和T-茎之间的核苷酸接头区域中的替代序列，可以部分恢复活性。类似物之一的D-和T-环中的点突变完全消除了白细胞化。在这种极简的变体中将A73更改为C73也废除了亮氨酰化作用，从而证实了鉴别基是主要的识别元素。这些结果表明，tRNA leu核心区域的三级相互作用对于LeuRS的识别至关重要。用LeuRS突变酶和每种可氨基酰化的tRNAleu类似物进行的水解编辑测定表明，可变环与酶的编辑位点相互作用。;还研究了在没有核糖体成分的情况下，氨基酰化RNA可能参与肽键形成的几种途径。测试了5'-亮氨酰-腺苷酸酐形成二肽的潜力。通过薄层色谱法检测Leu-Leu二肽和亮氨酸多肽。公开发表的声称，在存在氨基酰化的tRNA的情况下可以形成二肽，并详细研究了相应的mRNA模板，Mg 2+和用作催化剂的Ala-His二肽。使用[14C]-亮氨酸-tRNA leu和聚-AUUU mRNA，以及其他催化剂或底物重复进行已发表的反应。尽管在反应过程中形成了两种不确定的副产物，但在Ala-His的存在下Leu-Leu二肽合成并未发生。未知产物1显示为通过乙醇分解形成的亮氨酰基-乙基酯。未知产物2被鉴定为腺苷-亮氨酸。这些结果强烈表明，已发表的声明可能是虚假的。

著录项

  • 作者

    Larkin, Deana Christine.;

  • 作者单位

    University of Houston.;

  • 授予单位 University of Houston.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2001
  • 页码 138 p.
  • 总页数 138
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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