Role of the Universally Conserved 3'-CCA And Synthetase Recognition Determinants in tRNA Function

普遍保守的 3-CCA 和合成酶识别决定因素在 tRNA 功能中的作用

• 批准号: 9513932
• 负责人: Jack Horowitz
• 金额: $ 33.61万
• 依托单位: Iowa State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513932&HistoricalAwards=false
• 关键词: Role; Universally; Conserved; CCA; Synthetase

9513932 Horowitz The long-range goals of this research are to define structural features of transfer RNA essential for its function in protein synthesis. Focus will be on E. coli tRNA Val and its interaction with its cognate aminoacyl-tRNA synthetase. Methods of tRNA gene manipulation, site-directed mutagenesis, and in vitro transcription will be used to introduce modifications into E. coli tRNA Val and examine their effects on function, primarily by examining in vitro aminoacylation kinetics and polypeptide synthesis. a. Role of the universally conserved 3'-CCA sequence in tRNA function. Recent results have shown that E. coli tRNA Val can function actively in aminoacylation and polypeptide synthesis despite alterations in the universally conserved 3'-CCA end. To further explore the functional role of the 3'-CCA terminus the following will be done: (1) Functional groups in the 3' terminal sequence of tRNA essential for aminoacylation will be examined by site-specific incorporation of base analogs. (2) Role of 3'-CCA in misacylation and editing by valyl-tRNA synthetase will be investigated. (3) Role of 3' terminal nucleotides in polypeptide synthesis will be investigated. Although the variants of tRNA Val termination in 3' pyrimidines are readily aminoacylated, they function poorly in polypeptide synthesis. The step(s) of polypeptide synthesis inhibited by pyrimidine substituents at the 3' end of tRNA Val will be identified. (4) Ribosome recognition of the 3' end will be investigated using 3'-end mutants of tRNA. Val (5) Specific requirement for 3'-CCA in tRNAs other than tRNA Val will be examined. b. Recognition of E. coli tRNA Val by valyl-tRNA synthetase. The recognition of tRNA Val by its cognate synthetase will be further investigated and the synthetase itself will be further characterized. These studies will include the following: (1) Efforts will continue to define the complete set of sequence and structural elements essential for tRNA Val recognition by valyl-tRNA syn thetase. Features thus far identified as necessary for tRNA Val recognition will be inserted into other tRNAs to assess effects of sequence context on valine-accepting specificity and to identify possible negative determinants (antideterminants) that interfere with synthetase recognition. (2) Details of synthetase/tRNA interactions can best be resolved by structure determination of the complex. Large quantities of purified tRNA Val and valyl-tRNA synthetase are available and in collaboration with a crystallographer the structure of valyl-tRNA synthetase and its complexes will be determined. (3) The aminoacylation fidelity, hydrolytic editing, and other functions of an active truncated mutant of valyl-tRNA synthetase, which have recently been isolated, cloned, and overexpressed, will be characterized. %%% Transfer RNAs (tRNA) play an important role in determining the accuracy and efficiency of protein biosynthesis. These molecules effectively translate the sequence of 3-nucleotide codes determined by the genetic material into the linear incorporation of amino acids into proteins in their proper order. All tRNAs contain a universally conserved code (3'-CCA) at one end of the molecule which implicates a specific function. Interestingly, base changes within this sequence had no effect on protein synthesis. The proposed research will explore the functional role of the 3'-CCA sequence by evaluating the effects of altering chemical(functional) groups within this sequence on aminoacylation reaction kinetics as well as on recognition by the specific tRNA synthetase which mediates this reaction. The structure of the synthetase enzyme and its complexes with tRNA and other substrates will be obtained by X-ray crystallography. ***

9513932 Horowitz本研究的长期目标是确定其在蛋白质合成中的功能所必需的转运RNA的结构特征。重点将放在E。coli tRNA瓦尔及其与同源氨酰-tRNA合成酶的相互作用。tRNA基因操作、定点突变和体外转录的方法将被用于将修饰引入E。coli tRNA瓦尔，并主要通过检查体外氨酰化动力学和多肽合成来检查它们对功能的影响。 a.普遍保守的3 '-CCA序列在tRNA功能中的作用。 最近的研究结果表明，E. colitRNA瓦尔在氨基酸酰化和多肽合成中具有活性，尽管普遍保守的3 '-CCA末端发生了改变。为了进一步探索3 '-CCA末端的功能作用，将进行以下工作：（1）通过碱基类似物的位点特异性掺入来检查tRNA的3'末端序列中对于氨酰化必需的官能团。(2)将研究3 '-CCA在缬氨酰-tRNA合成酶的错酰化和编辑中的作用。 (3)3'末端核苷酸在多肽合成中的作用将被研究。 虽然tRNA的瓦尔末端在3'嘧啶上的变体很容易被氨酰化，但它们在多肽合成中的功能很差。 tRNA瓦尔3'端嘧啶取代基抑制的多肽合成步骤 将被识别。(4)将使用tRNA的3 '端突变体研究3'端的核糖体识别。瓦尔（5）将检查除tRNA瓦尔以外的tRNA中对3 '-CCA的特定要求。 B.对E. colitRNA通过缬氨酰-tRNA合成酶合成瓦尔。将进一步研究tRNA瓦尔被其同源合成酶识别，并进一步表征合成酶本身。 这些研究将包括以下内容：（1）将继续努力确定缬氨酰-tRNA合成酶识别tRNA瓦尔所必需的完整序列和结构元件。将迄今为止鉴定为tRNA瓦尔识别所必需的特征插入到其他tRNA中，以评估序列背景对缬氨酸接受特异性的影响，并鉴定干扰合成酶识别的可能的负决定簇（反决定簇）。(2)合成酶/tRNA相互作用的细节可以通过复合物的结构测定来最好地解决。 大量的纯化的tRNA瓦尔和缬氨酰-tRNA合成酶是可用的，并且与晶体学家合作，将确定缬氨酰-tRNA合成酶及其复合物的结构。(3)氨酰化保真度，水解编辑，和其他功能的活性截短突变的缬氨酰-tRNA合成酶，最近已被分离，克隆，和过表达，将其特征在于。 转移RNA（tRNA）在决定蛋白质生物合成的准确性和效率方面起着重要作用。 这些分子有效地将由遗传物质决定的3核苷酸密码序列翻译成氨基酸以其适当顺序线性掺入蛋白质中。 所有的tRNA在分子的一端都含有一个普遍保守的密码（3 '-CCA），它暗示了一种特定的功能。 有趣的是，该序列中的碱基变化对蛋白质合成没有影响。 拟议的研究将通过评估改变该序列内的化学（功能）基团对氨酰化反应动力学的影响以及对介导该反应的特异性tRNA合成酶的识别来探索3 '-CCA序列的功能作用。 合成酶及其与tRNA和其他底物的复合物的结构将通过X射线晶体学获得。 ***