Mechanisms of translation initiation on cellular IRES e*

描述（由申请人提供） 这项研究将主要在俄罗斯的莫斯科州立大学与William梅里克博士合作进行，作为在他的实验室进行的研究的延伸。该项目涉及真核细胞的细胞内部核糖体进入位点（IRES）的研究。细胞IRES使用识别起始密码子的非典型机制指导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不同的是，没有一种细胞IRES的功能机制是已知的。它们的二级结构与已知病毒IRES的二级结构不相似，并且它们之间也不相似。该提案旨在剖析一些细胞IRES的翻译起始的分子机制，主要使用强大的现代技术，即从纯化组分组装翻译起始复合物，并结合足印测定。在本研究中，选择了三种细胞IRES，基于它们在无细胞系统中的有效翻译，因此推测其功能对翻译起始组分的要求更简单：编码NRF（NF-κ B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IRES。将使用纯化的翻译组分组装这些IRES上的翻译起始复合物，并确定起始因子要求。这些IRES的边界和最重要的功能位点将通过缺失分析来定义。将测试产生的每种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。将通过足迹法确定与IRES关键功能位点结合的翻译组分。

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.