Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

核糖体组装的质量控制 - 调节蛋白的功能

• 批准号: 9161382
• 负责人: Katrin Karbstein
• 金额: $ 5.72万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9161382
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Binding; Biochemical; Biochemistry; Bypass; Cells; Communication; Development; Diamond-Blackfan anemia; Disease; Dissociation; Elongation Factor; Ensure; Failure; Funding; Genetic; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hybrids; Hydrolysis; In Vitro; Incidence; Kinetics; Link; Malignant Neoplasms; Maps; Messenger RNA; Molecular; Pathway interactions; Patients; Polyribosomes; Proteins; Quality Control; Reaction; Reagent; Ribosomal Proteins; Ribosomes; Role; Sedimentation process; Staging; Structure; Syndrome; Testing; Tissues; Transfer RNA; Translating; Translations; Work; Yeasts; blocking factor; chromosome 5q loss; eukaryotic initiation factor-5B; factor A; genetic regulatory protein; insight; mutant; public health relevance; reconstitution; release factor; research study; response; termination factor; translation factor

DESCRIPTION (provided by applicant): PROJECT SUMMARY Rapidly dividing cells must produce 2,000 new ribosomes every minute, and ensure that they are fully functional. Escape of incompletely assembled ribosomes into the translating pool has been suggested to underlie the high cancer incidence observed in patients suffering from Diamond Blackfan Anemia, 5q- syndrome and congenital asplenia. These considerations suggest that in healthy cells mechanisms must be in place to ensure that only correctly assembled ribosomes are released into the translating pool. In the previous funding period, we have discovered such a quality control mechanism, which uses the translational machinery to test the functionality of assembling 40S subunits: Pre-40S subunits are joined by mature 60S subunits in a reaction promoted by the translation factor eIF5B. Assembly factors dissociate from these 80S-like ribosomes, which do not produce protein, as they do not contain mRNA or tRNA. Release of assembling subunits into the translating pool is gated by the termination factors Rli1 and Dom34. However, exactly how progression of this cascade is linked to successful completion of functional tests is unknown, as is the mechanism by which assembly factors are released from 80S-like ribosomes. Here we address the hypothesis that assembly factors and translation factors cooperate to release assembly factors, and induce conformational changes in assembling ribosomes that are akin to those that underlie its function during translation. In Aim 1 we will dissect how the ATPase activity of the assembly factor Rio2 is used to trigger conformational changes in pre- 40S subunits that allow for joining of 60S subunits. In Aim 2 we will test how the translation factor eIF5B is used to release the assembly factor Rio2 from pre-40S subunits. In Aim 3 we will discern how the assembly factor Fap7 and the translation factor eEF2 cooperate to promote conformational changes used during translation for the translocation of tRNA and mRNA. These experiments take advantage findings and reagents produced during the last funding period, and use a unique combination of genetic, biochemical and structural experiments to address these fundamental questions.

描述（由申请人提供）： 快速分裂的细胞必须每分钟产生2,000个新的核糖体，并确保它们具有完整的功能。不完全组装的核糖体逃逸到翻译池中已被认为是在患有Diamond Blackfan贫血、5 q-综合征和先天性无脾的患者中观察到的高癌症发病率的基础。这些考虑表明，在健康的细胞中，必须有适当的机制来确保只有正确组装的核糖体被释放到翻译池中。在之前的资助期间，我们已经发现了这样一种质量控制机制，它使用翻译机制来测试组装40 S亚基的功能：在翻译因子eIF 5 B促进的反应中，前40 S亚基与成熟的60 S亚基连接。装配因子从这些80 S样核糖体中分离出来，这些核糖体不产生蛋白质，因为它们不包含mRNA或tRNA。组装亚基释放到翻译池是由终止因子Rli 1和Dom 34门控。然而，这种级联反应的进展与成功完成功能测试的确切联系尚不清楚，组装因子从80 S样核糖体释放的机制也是未知的。在这里，我们解决的假设，组装因子和翻译因子合作释放组装因子，并诱导组装核糖体的构象变化，类似于那些在翻译过程中的功能。在目的1中，我们将剖析组装因子Rio 2的ATP酶活性如何用于触发前40 S亚基的构象变化，从而允许60 S亚基的连接。在目标2中，我们将测试翻译因子eIF 5 B如何用于从前40 S亚基释放组装因子Rio 2。在目标3中，我们将了解组装因子Fap 7和翻译因子eEF 2如何合作促进翻译过程中用于tRNA和mRNA移位的构象变化。这些实验利用了上一个资助期内的发现和试剂，并使用遗传、生物化学和结构实验的独特组合来解决这些基本问题。