Molecular determinants of A-site mRNA cleavage during ribosome pausing

描述（由申请人提供）： 该研究项目的长期目标是了解细胞如何在蛋白质合成过程中识别和回收无活性的核糖体。并非所有启动翻译的核糖体都能够完成全长蛋白质的合成。在许多情况下，不完全的蛋白质合成是由于“非终止”mRNA，其是缺少框内终止密码子的截短转录物。在细菌中，被困在不间断mRNA的34端的核糖体被tmRNA（转移信使RNA）质量控制系统“拯救”。尽管tmRNA在真细菌中普遍存在，但由于存在平行的不依赖于tmRNA的核糖体拯救途径，它可以从大肠杆菌细胞中删除。然而，这两种途径在E.大肠杆菌，证明核糖体拯救是这些细胞，可能是所有细胞的基本功能。该建议集中在围绕核糖体拯救的翻译质量控制的三个基本方面。首先，我们试图确定A位点mRNA切割和核糖体拯救之间的功能关系。A位点切割是一种新的RNA酶活性，其截断停滞核糖体的A位点密码子中的mRNA，从而产生不终止的mRNA。将使用生物化学和分子遗传学方法来鉴定A位点核酸酶。A位点核酸酶的鉴定对于确定其活性是否在tRNA介导的或非tRNA依赖的核糖体拯救中发挥功能性作用是必要的。其次，我们将描述最近发现的tRNA独立的救援途径，这似乎是由YhdL介导的。将使用体外和体内方法研究YhdL的生化功能和调节。将鉴定YhdL核糖体拯救活性的谱，并确定其在新生链释放中的作用。最后，DnaK在tRNA介导的核糖体拯救中的作用将被机械地定义。将使用分子遗传学和药理学方法消除DnaK活性，并评估对tmRNA标记的影响。定义的体外翻译系统将用于确定DnaK是否通过其共翻译伴侣活性发挥其作用。

DESCRIPTION (provided by applicant): The long-term objective of this research project is to understand how cells identify and recycle inactive ribosomes during protein synthesis. Not all ribosomes that initiate translation are able to complete synthesis of full-length proteins. In many instances, incomplete protein synthesis is due to "non-stop" mRNAs, which are truncated transcripts lacking in-frame stop codons. In bacteria, ribosomes trapped at the 34-ends of non-stop mRNA are "rescued" by the tmRNA (for transfer-messenger RNA) quality control system. Although tmRNA is ubiquitous throughout the eubacteria, it can be deleted from Escherichia coli cells due to the presence of a parallel tmRNA-independent ribosome rescue pathway. However, both pathways cannot be disrupted in E. coli, demonstrating that ribosome rescue is an essential function for these and probably all cells. This proposal focuses on three fundamental aspects of translational quality control that revolve around ribosome rescue. First, we seek to determine the functional relationship between A-site mRNA cleavage and ribosome rescue. A- site cleavage is a novel RNase activity that truncates mRNA in the A-site codon of stalled ribosomes, thereby producing non-stop mRNA. Biochemical and molecular genetic approaches will be used to identify the A-site nuclease. Identification of the A-site nuclease is necessary to determine whether the activity plays a functional role in either tmRNA-mediated or tmRNA-independent ribosome rescue. Second, we will characterize the recently identified tmRNA-independent rescue pathway, which appears to be mediated by YhdL. The biochemical function and regulation of YhdL will be investigated using in vitro and in vivo approaches. The spectrum of YhdL ribosome rescue activity will be identified, and its role in nascent chain release determined. Finally, the role of DnaK in tmRNA-mediated ribosome rescue will be mechanistically defined. DnaK activity will be ablated using molecular genetic and pharmacological approaches and the effects on tmRNA tagging assessed. A defined in vitro translation system will be used to determine whether DnaK exerts its effects by virtue of its co-translational chaperone activity.