Mechanisms of translation initiation on cellular IRES of eukaryotic cells

真核细胞 IRES 翻译起始机制

基本信息

批准号：
7169883
负责人：
WILLIAM C. MERRICK
金额：
$ 3.1万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-12-15 至 2008-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7169883
关键词：
Active Sites Antibodies Apoptosis Binding Binding Proteins Biological Assay Bite Cell Differentiation process Cell division Cell physiology Cell-Free System Cells Cis-Acting Sequence Codon Nucleotides Collaborations Complex Electrophoretic Mobility Shift Assay Eukaryotic Cell Event Evolution Gel Gene Expression Genetic Translation Genome Grant Homologous Gene Human Initiator Codon Internal Ribosome Entry Site Laboratories Lead Maps Mediating Messenger RNA Modification Molecular Monitor Moscow NF-kappa B Numbers Nutritional Pathway interactions Peptide Initiation Factors Polyribosomes Principal Investigator Protein Biosynthesis Protein Isoforms Proteins Publications RNA Binding RNA Helicase Recombinants Research Retrotransposon Ribosomes Russia Scanning Site Standards of Weights and Measures Stimulus Stress Structure System Techniques Temperature Testing Trans-Activators Translation Initiation Translations United States National Institutes of Health Universities Viral Yeasts base cell type deletion analysis helicase mutant parent grant programs response yeast two hybrid system

项目摘要

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）这项研究将主要在莫斯科州立大学与威廉·梅里克（William Merrick）博士合作，以扩展其实验室的研究。该项目与真核细胞的细胞内核糖体进入位点（IRESS）有关。细胞IRESS直接mRNA翻译使用了启动起始密码子识别的非典型机制。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。但是，与某些病毒性IRESS不同，没有细胞IRESS是已知功能的机制。它们的二级结构与已知的病毒率的结构不同，并且在彼此之间不同。该建议旨在剖析某些细胞IRES的翻译启动的分子机制，主要是使用强大的现代翻译起始组装技术与纯化的成分结合了托管分析。 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。这些IRESS上的翻译启动络合物将使用纯化的翻译组件组装，并确定启动因子要求。这些IRESS和最重要的功能位点的边界将通过删除分析来定义。产生的每个突变体将测试其形成翻译起始复合物并维持转染细胞中的IRES活性的能力。与IRESS关键功能位点结合的翻译成分将由足迹确定。