Elucidating premature translation termination in Cystic Fibrosis

项目总结 囊性纤维化的一个主要原因是囊性纤维化中的提前终止密码子(PTCs) 跨膜电导调节因子(CFTR)基因。抑制PTC处的翻译终止--即PTC 通读-恢复全长CFTR蛋白可能是一种治疗策略。然而，目前的PTC通读 CF的候选药物是有毒的(如氨基糖苷类)或无效的(如阿塔鲁仑)。PTC通读的疗效 取决于PTC的翻译终止效率。因此，操控病毒的分子机制 CFTRPTC终止以降低效率可能会提高PTC的通读效率。然而，针对这种情况的战略 在缺乏对CFTRPTC翻译终止的详细了解的情况下，操作是有限的。 在目前的正常终止模型中，真核释放因子1和3形成一个复合体(eRF1·eRF3) 这会从核糖体中释放一种新合成的蛋白质。ERF1识别四核苷酸终止密码子 开放阅读框的末端，并催化多肽-tRNA的水解。Poly-A结合蛋白(PABP)，它 结合在mRNA的3‘端，募集eRF3，提高终止效率。然而，目前还不清楚是如何 PABP、eRF1、eRF3和四核苷酸终止密码子识别PTC，从而产生截短的CFTR蛋白。 这一建议的目的是确定翻译终止的生化机制和结构机制。 在Andrei Korostelev博士(生化和结构翻译机制专家)的指导下， 艾伦·雅各布森(提前终止翻译和PTC通读方面的专家)，Phillip Zamore博士(RNA 生物化学家)、陈煦博士(低温EM仪器专家)和尼古拉斯·格里戈里夫博士(低温EM方法专家 开发)，释放测试将被优化以研究由 真核释放因子和集成时间分辨(完整)冷冻-EM将被用来捕获结构 酶反应的中间体。目标1将使用定义的哺乳动物翻译系统来测量 终止密码子环境、eRF1·eRF3和PABP对CFTR终止效率(kcat/Km)的单独影响 PTCS和真正的终止密码子。AIM 2将可视化显示核糖体如何在其 使用整个低温EM的自然序列上下文。在多个时间点收集数据将确定 终止过程中mRNA序列、eRF1·eRF3和PABP的构象变化及相互作用。 为了揭示CFTRPTCs的终止机制，结构和它们的进展中间体将是 与记录在真实cftr终止密码子上的基因进行比较。如果成功，这项研究将揭示关键分子 CFTR PTC终止的决定因素，并可能为诱导CF治疗PTC通透的策略提供参考。

PROJECT SUMMARY A leading cause of Cystic Fibrosis (CF) is premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Suppression of translation termination at PTCs—i.e. PTC readthrough—to restore full-length CFTR protein may be a treatment strategy. Yet, current PTC readthrough drug candidates for CF are toxic (e.g. aminoglycosides) or ineffective (e.g. ataluren). Efficacy of PTC readthrough depends on efficiency of translation termination at the PTC. Thus, manipulating the molecular mechanisms of CFTR PTC termination to lower efficiency may improve PTC readthrough efficacy. However, strategies for such manipulations are limited in the absence of a detailed understanding of translation termination on CFTR PTCs. In the current model for normal termination, eukaryotic Release Factors 1 and 3 form a complex (eRF1•eRF3) that releases a newly synthesized protein from the ribosome. eRF1 recognizes a tetra-nucleotide stop codon at the end of an open reading frame, and catalyzes peptidyl-tRNA hydrolysis. Poly-A binding protein (PABP), which binds at 3′ ends of mRNA, recruits eRF3 and enhances termination efficiency. However, it remains unclear how PABP, eRF1, eRF3, and the tetra-nucleotide stop codon recognize the PTC to produce truncated CFTR protein. The goal of this proposal is to determine the biochemical and structural mechanism of translation termination. With guidance from Dr. Andrei Korostelev (expert in biochemical and structural mechanisms of translation), Dr. Allan Jacobson (expert in premature translation termination and PTC read-through), Dr. Phillip Zamore (RNA biochemist), Dr. Chen Xu (cryo-EM instrumentalist), and Dr. Nikolaus Grigorieff (expert in cryo-EM method development), release assays will be optimized to study the efficiency of translation termination mediated by eukaryotic release factors, and ensemble time-resolved (ENTIRE) cryo-EM will be used to capture structural intermediates of enzymatic reactions. Aim 1 will use defined mammalian translation systems to measure the individual effects of stop codon context, eRF1•eRF3, and PABP on the termination efficiencies (kcat/KM) of CFTR PTCs and the true stop codon. Aim 2 will visualize how the ribosome terminates on CFTR PTC G542X in its natural sequence context using ENTIRE cryo-EM. Collecting data at multiple time points will identify conformational changes and interactions between mRNA sequence, eRF1•eRF3, and PABP during termination. To reveal the termination mechanism on CFTR PTCs, structures and their progression intermediates will be compared with those recorded on the true CFTR stop codon. If successful, this study will reveal key molecular determinants of CFTR PTC termination, and may inform strategies to induce PTC readthrough for CF treatment.