Mechanism of Eukaryotic Translation Termination

描述(由申请人提供)： 在真核生物中，eRF1识别三个翻译终止密码子，并介导新生多肽链的释放。辅助因子eRF3以依赖于GTP的方式辅助终止过程。在过去的几年里，我们对eRF1如何识别终止密码子和促进多肽链释放的了解有所增加，但仍有许多工作要做。相比之下，几乎没有人了解eRF3对GTP的水解如何在终止过程中起到帮助作用。与原核生物不同，eRF3是酵母中的一个必不可少的基因。此外，eRF3不仅与eRF1相互作用，而且还与参与mRNA功能和代谢的其他蛋白质相互作用，包括PolyA结合蛋白和Upflp、Upf2p和Upf3p。这表明eRF3不仅在翻译终止过程中起作用，而且在终止过程中还与其他相关的细胞功能，如mRNA稳定性、NMD，以及可能的翻译启动有关。为了更好地了解eRF1和eRF3在翻译终止中的作用，我们提出了以下具体目标：具体目标1)：确定eRF3在翻译终止中的功能。我们将把eRF3的突变分析与体外和体内功能分析结合起来，以确定eRF3在这一过程中的作用。具体目标2)：确定eRF1和核糖体如何影响eRF3在翻译终止中的功能。我们将使用功能分析来确定这些因素如何刺激eRF3的GTPase活性。具体目标3)：确定eRF1的磷酸化和/或甲基化是否在翻译终止过程中调节其功能。我们将使用质谱学来表征eRF1的翻译后修饰。然后，我们将研究磷酸化如何影响eRF1功能，并确定在真核生物中是否高度保守的GGQ基序甲基化。具体目标4)：确定Pst21p和slhlp这两个新因素如何影响翻译终止的效率。我们将研究这些因子是否直接与eRF1、eRF3或Upflp相互作用，并研究这些蛋白质中的功能基序，这些功能基序可能为它们的细胞功能提供线索。

DESCRIPTION (provided by applicant): In eukaryotic organisms, eRF1 recognizes the three translation termination codons and mediates the release of nascent polypeptide chains. The accessory factor eRF3 assists the termination process in a GTP-dependent manner. Over the last few years, our understanding of how eRF1 recognizes stop codons and facilitates polypeptide chain release has increased, but much work remains to be done. In contrast, little has been done to understand how GTP hydrolysis by eRF3 assists in the termination process. Unlike prokaryotes, eRF3 is an essential gene in yeast. Furthermore, eRF3 has been shown to interact not only with eRF1, but also with other proteins involved in various aspects of mRNA function and metabolism, including poly(A)-binding protein and Upflp, Upf2p, and Upf3p. This suggests that eRF3 may act not only in translation termination, but also couple the termination process to other related cellular functions such as mRNA stability, NMD, and possibly translation initiation. To better understand how eRF1 and eRF3 function in translation termination, we propose the following Specific Aims: Specific Aim 1): Determine the function of eRF3 in translation termination. We will couple a mutational analysis of eRF3 to in vitro and in vivo functional assays to determine the role of eRF3 in this process. Specific Aim 2): Determine how eRF1 and the ribosome influence eRF3 function in translation termination. We will use functional assays to determine how these factors stimulate the GTPase activity of eRF3. Specific Aim 3): Determine whether phosphorylation and/or methylation of eRF1 modulates its function during translation termination. We will use mass spectrometry to characterize the post-translational modification of eRF1. We will then examine how phosphorylation influences eRF1 function, and determine whether the highly conserved GGQ motif is methylated in eukaryotes. Specific Aim 4): Determine how two new factors, Pst21p and Slhlp, influence the efficiency of translation termination. We will examine whether these factors interact directly with eRF1, eRF3, or Upflp, and examine functional motifs in these proteins that may provide clues to their cellular function.