Function of the TRAMP Complex at the Molecular Level

描述(由申请人提供)：拟议研究的总体目标是获得由TRAMP(Trf4/Air2/MTR4多聚腺苷基化)和核外切体形成的多组分复合体的详细机制了解。这种游动-外切体机制在真核基因表达的调控中起着关键作用。TRAMP由三个在真核生物中高度保守的亚基组成，即一个非规范的聚(A)聚合酶、一个锌指关节蛋白和一个RNA解旋酶。真核核外体由11个单位组成，它们在真核生物中也高度保守，包括两个功能性的3‘到5’核酸酶：Rrp6p和Rrp44p。TRAMP用短的Poly(A)尾巴标记RNA，并解析RNA的二级结构，而外体则降解由TRAMP标记的RNA。尽管在RNA代谢中起着关键作用，但在RNA加工过程中，TRAMP-外切体机制的组件如何协调它们的功能，以及RNA底物是如何识别的，目前还不清楚。在我们的提案中，我们使用生化手段来解决这些问题。利用重组的TraMP-exosome系统，我们将描述哪些exosome和traMp的成分和活性对TraMP刺激exosome至关重要，以及exosome对traMP功能的影响。为了了解在单个反应步骤水平上由浮动外切体机器对RNA的加工，我们将为反应设计一个机械性的、定量的框架。然后，我们将研究TRAMP-exosome机制如何识别RNA靶标。为此，我们将调查低修饰的tRNAiMet如何与正确修改的tRNAiMet区分开来，TRAMP-Exosome机制如何展开RNA，以及RNA长度和二级结构是否以及如何影响TRAMP-Exosome机制。我们期待我们的研究能为我们提供一个新的水平，从机制上理解外显体的机制，并对核RNA新陈代谢的分子基础有新的、有意义的见解。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to obtain detailed mechanistic understanding of the multi-component complex formed by TRAMP (Trf4/Air2/Mtr4 Polyadenylation) and the nuclear exosome. This TRAMP-exosome machinery plays key roles in the regulation of eukaryotic gene expression. TRAMP consists of three subunits that are highly conserved in eukaryotes, a non-canonical poly(A) polymerase, a Zn-knuckle protein, and an RNA helicase. The eukaryotic nuclear exosome consists of eleven units, which are also highly conserved in eukaryotes, and include two functional 3' to 5' nucleases, Rrp6p and Rrp44p. TRAMP marks RNAs with short poly(A) tails, and resolves RNA secondary structures, while the exosome degrades the RNAs marked by TRAMP. Despite pivotal roles in RNA metabolism, it is not understood how the components of the TRAMP-exosome machinery coordinate their functions during RNA processing, and how RNA substrates are identified. In our proposal, we address these problems using biochemical means. Using a reconstituted TRAMP-exosome system, we will delineate which exosome and TRAMP components and activities are critical for the exosome stimulation by TRAMP, and for effects of the exosome on TRAMP function. To understand RNA processing by the TRAMP-exosome machinery at the level of individual reaction steps, we will devise a mechanistic, quantitative framework for the reaction. We will then examine how the TRAMP-exosome machinery identifies RNA targets. To this end we will investigate how hypomodified tRNAiMet is distinguished from correctly modified tRNAiMet, how the TRAMP-exosome machinery unfolds the RNAs, and whether and how RNA length and secondary structure affect the TRAMP-exosome machinery. We anticipate our studies to provide a new level of mechanistic understanding of the TRAMP-exosome machinery, and novel, significant insight into the molecular basis of nuclear RNA metabolism.