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

基本信息

批准号：
8696139
负责人：
Katrin Karbstein
金额：
$ 47.07万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
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/8696139
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

项目摘要

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）：项目摘要迅速分裂的细胞必须每分钟产生2,000个新核糖体，并确保它们具有完全的功能。已经建议将不完全组装的核糖体逃入翻译池中，这是在患有钻石黑芬贫血，5q-综合征和先天性阿斯普氏症患者中观察到的高癌症发生率的基础。这些考虑因素表明，在健康细胞中必须建立机制，以确保仅正确组装的核糖体被释放到翻译池中。在上一个资金期间，我们发现了这种质量控制机制，该机制使用翻译机制测试组装40S亚基的功能：40S前的亚基与翻译因子EIF5B促进的反应中成熟的60S亚基相连。组装因子与这些80s样核糖体分离，这些核糖体不产生蛋白质，因为它们不包含mRNA或tRNA。终止因子RLI1和DOM34释放组装亚基在翻译池中。但是，该级联反应与成功完成功能测试的恰好如何相关，而从80年代样核糖体释放组装因子的机制也未知。在这里，我们解决了以下假设：组装因子和翻译因子合作以释放装配因子，并在组装核糖体中构象变化，类似于在翻译过程中其功能的基础的核糖体。在AIM 1中，我们将剖析装配因子RIO2的ATPase活性如何用于触发40S前亚基的构象变化，以允许连接60s亚基。在AIM 2中，我们将测试翻译因子EIF5B如何使用40年前的亚基释放装配因子RIO2。在AIM 3中，我们将辨别组装因子FAP7和翻译因子EEF2如何配合，以促进转化过程中用于转移tRNA和mRNA的构象变化。这些实验利用了最后一个融资期间产生的优势发现和试剂，并利用遗传，生化和结构实验的独特组合来解决这些基本问题。