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威廉·梅里克博士合作进行，作为其实验室正在进行的研究的延伸。该项目涉及真核细胞的细胞内部核糖体进入位点(IRESS)的研究。细胞内的IRESS使用非典型的识别起始密码子的机制来直接翻译mRNA。它们参与了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IRESS不同的是，没有一个细胞IRESS的功能机制是已知的。它们的二级结构与已知病毒IRESS的二级结构不同，而且它们之间也不同。该提案旨在主要利用一种强大的现代技术，即从纯化的成分中组装翻译起始复合体，并结合脚印分析，来剖析一些细胞IRESS的翻译起始的分子机制。在本研究中，基于它们在无细胞系统中的高效翻译而选择了三个细胞内IREs，因此推测对翻译起始成分执行其功能的要求更简单：编码NRF(核因子-KB抑制因子)的mRNA的IRES，来自从人反转录转座子L1转录的天然双顺反子mRNA的跨顺反子IRES，以及指导人HSP 70mRNA的翻译。这些IRESS上的翻译起始复合体将使用纯化的翻译成分进行组装，并将确定起始因子要求。这些IRESS和最重要的功能位点的边界将通过缺失分析来确定。每个产生的突变体都将被测试其在转基因细胞中形成翻译起始复合体和保持IRES-活性的能力。与IRESS关键功能位点结合的翻译成分将通过足迹确定。