THE MECHANISMS OF EUKARYOTIC TRANSLATION TERMINATION AND RIBOSOMAL RECYCLING

描述(申请人提供)：真核终止是两个释放因子eRF1和eRF3之间复杂的功能相互作用的结果，其中eRF3的GTP水解结合密码子识别和eRF1的肽-tRNA水解。终止后复合体(Post-TCS)的循环是由ATP结合盒家族的重要成员ABCE1和eRF1共同作用促进的，eRF1在肽释放后仍与后TCS相关。因此，eRF1参与了蛋白质合成、终止和核糖体循环的两个连续阶段。在某些情况下，后TCS不经历完全的循环，终止之后是对相同的mRNA的重新启动，通常在终止密码子的下游。这主要发生在短开放阅读框(ORF)的翻译之后，而长ORF后的重新启动很少见，似乎仅限于几个病毒家族。终止/再循环还与另外两个重要的过程有关，即通过No-Go和无意义介导的衰变(分别为NGD和NMD)来监测mRNA。NGD针对的是延伸复合体(EC)通过稳定的二级结构而停滞的mRNAs。它是由停滞的核糖体附近的内切核糖体启动的，S被eRF1和eRF3类似物Pelota和Hbs1刺激，然后与ABCE1一起解离内皮细胞，而不需要事先释放肽。NMD针对的是含有提前终止密码子(PTCs)的异常mRNAs，PTCs是由异常剪接、突变或转录错误引起的。含有PTC的mRNAs的识别和靶向降解是由保守的效应分子介导的，包括UPF1(一种死盒RNA解旋酶)、UPF2、UPF3B和UPF1激酶SMG1。UPF1与eRF1、eRF3、UPF2、UPF3B和SMG1相互作用，在此过程中发挥关键作用。有人认为，在PTC滞留的核糖体是由SURF(SMG1，UPF1，eRF1/3)监视复合体识别的，UPF1的招募也会损害终止。拟议的研究将基于从单个纯化的翻译成分体外重建蛋白质合成的所有阶段(启动、延伸、终止和核糖体回收)的方法。在目标1中，我们建议研究与该过程不同阶段相对应的核糖体循环和终止复合体的结构，并表征顺式作用的mRNA元件和反式作用因子是如何调节终止的。在目标2中，我们将使用我们的体外重组系统总结不同的重新启动模式，以确定它们的因素需求和机制。在目标3中，我们将定义ABCE1/Pelota介导的停滞的核糖体延长复合体解离的要求，并将尝试定位NGD中内核溶解切割的位置，并确定负责的核酸酶。目的4将致力于研究UPF1在翻译过程中对各个步骤的影响，这取决于它的磷酸化状态以及UPF2和UPF3辅助因子的存在。

DESCRIPTION (provided by applicant): Eukaryotic termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition and peptidyl-tRNA hydrolysis by eRF1. Recycling of post-termination complexes (post-TCs) is promoted by the concerted action of ABCE1, an essential member of ATP-binding cassette family of proteins, and eRF1, which remains associated with post- TCs after peptide release. eRF1 thus participates in two successive stages of protein synthesis, termination and ribosome recycling. In some cases post-TCs do not undergo complete recycling, and termination is instead followed by reinitiation on the same mRNA, usually downstream of the stop codon. This mostly occurs after translation of short open reading frames (ORFs), whereas reinitiation after long ORFs is rare and appears to be limited to several virus families. Termination/recycling are also associated with two other important processes, mRNA surveillance by No-Go and nonsense-mediated decay (NGD and NMD, respectively). NGD targets mRNAs on which elongation complexes (ECs) are stalled by e.g. stable secondary structure. It is initiated by endonucleolytic cleavage in the vicinity of stalled ribosomes, which s stimulated by eRF1 and eRF3 paralogues Pelota and Hbs1 that together with ABCE1 can then dissociate ECs without prior peptide release. NMD targets aberrant mRNAs containing premature termination codons (PTCs), which are introduced by aberrant splicing, mutation or transcription errors. Recognition and targeting for degradation of PTC-containing mRNAs is mediated by conserved effectors, including UPF1 (a DEAD-box RNA helicase), UPF2, UPF3B and the UPF1 kinase SMG1. UPF1 interacts with eRF1, eRF3, UPF2, UPF3B and SMG1, and plays a key role in the process. It was suggested that ribosomes arrested at PTCs are recognized by a SURF (SMG1, UPF1, eRF1/3) surveillance complex, and that recruitment of UPF1 also impairs termination. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosoma recycling) from individual purified translational components. In Aim 1, we propose to investigate the structure of ribosome recycling and termination complexes corresponding to different stages in the process, and to characterize how termination is regulated by cis-acting mRNA elements and trans-acting factors. In Aim 2 we will recapitulate different modes of reinitiation using our i vitro reconstituted system to determine their factor requirements and mechanisms. In Aim 3 we will define the requirements for ABCE1/Pelota-mediated dissociation of stalled ribosomal elongation complexes, and will undertake an attempt to localize the site of endonucleolytic cleavage in NGD and to identify the responsible nuclease. Aim 4 will be devoted to investigation of the influence of UPF1, depending on its phosphorylation status and the presence of UPF2 and UPF3 cofactors, on individual steps in the translation process.