Mechanisms of translation initiation on cellular IRES e*

细胞 IRES e* 的翻译起始机制

• 批准号: 6999717
• 负责人: WILLIAM C. MERRICK
• 金额: $ 3.19万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999717
• 关键词: DNA footprinting; binding proteins; binding sites; gel mobility shift assay; gene deletion mutation; gene expression; genetic translation; heat shock proteins; international cooperation; messenger RNA; microarray technology; molecular genetics; nuclear factor kappa beta; protein isoforms; protein protein interaction; ribosomes; site directed mutagenesis; translation factor; transposon /insertion element; yeast two hybrid system; yeasts

DESCRIPTION (provided by applicant) This research will be done primarily in Russia at Moscow State University in collaboration with Dr. William Merrick as an extension of the research being conducted in his laboratory. The project is concerned with studies of cellular internal ribosome entry sites (IRESs) of eukaryotic cells. The cellular IRESs direct mRNA translation using atypical mechanisms of recognition of the initiation start codon. They are involved in many important cellular processes and responses: genome evolution, cell-division, apoptosis, cell differentiation, response to nutritional stimuli etc. However, unlike some of the viral IRESs, for none of the cellular IRESs is the mechanism of functioning known. Their secondary structures do not resemble those of known viral IRESs and are dissimilar among themselves. The proposal aims to dissect molecular mechanisms of translation initiation for some of cellular IRESs mostly using a powerful modern technique of assembly of translation initiation complexes from purified components combined with the toeprint assay. For this study, three cellular IRESs were selected on the base of their efficient translation in a cell-free system and therefore presumed simpler requirement for translation initiation components to perform their function: the IRES of the mRNA encoding NRF (NF-Kb repressing factor), the intercistronic IRES from a natural dicistronic mRNA transcribed from human retrotransposon L1, and that directing translation of human Hsp 70 mRNA. The translation initiation complexes on these IRESs will be assembled using purified translational components and the initiation factor requirements will be determined. The boundaries of these IRESs and the most important functional sites will be defined by deletion analysis. Each of the mutants produced will be tested for its ability to form translation initiation complexes and to maintain the IRES-activity in transfected cells. The translational components that bind to the critical functional sites of the IRESs will be determined by footprinting.

描述（由申请人提供） 这项研究将主要在俄罗斯莫斯科国立大学与 William Merrick 博士合作进行，作为其实验室研究的延伸。该项目涉及真核细胞的细胞内部核糖体进入位点（IRES）的研究。细胞 IRES 使用非典型的起始起始密码子识别机制指导 mRNA 翻译。它们参与许多重要的细胞过程和反应：基因组进化、细胞分裂、细胞凋亡、细胞分化、对营养刺激的反应等。然而，与一些病毒 IRES 不同，没有一个细胞 IRES 的功能机制是已知的。 。它们的二级结构与已知的病毒 IRES 的二级结构不同，并且它们之间也不同。该提案旨在剖析一些细胞 IRES 翻译起始的分子机制，主要使用强大的现代技术，将纯化成分组装成翻译起始复合物，并结合脚趾印迹测定。在本研究中，根据其在无细胞系统中的有效翻译，选择了三种细胞 IRES，因此推测翻译起始组件执行其功能的要求更简单：编码 NRF（NF-Kb 抑制因子）的 mRNA 的 IRES ，来自人逆转录转座子 L1 转录的天然双顺反子 mRNA 的顺反子间 IRES，以及指导人 Hsp 70 mRNA 翻译的顺反子 IRES。这些 IRES 上的翻译起始复合物将使用纯化的翻译组件进行组装，并确定起始因子的要求。这些 IRES 的边界和最重要的功能位点将通过删除分析来定义。将测试产生的每个突变体在转染细胞中形成翻译起始复合物和维持 IRES 活性的能力。与 IRES 关键功能位点结合的翻译组件将通过足迹法确定。