Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation
Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation
复制标题
DOI：
10.1016/j.jmb.2005.07.037
复制
发表时间：
2005-09-16
影响因子：
5.6
通讯作者：
Fasiolo, F
中科院分区：
文献类型：
--
作者：
Graindorge, JS;Rousselle, JC;Fasiolo, F
先前的研究表明，核核Tif6从新生的60s亚基释放发生在细胞质中，并且需要细胞质中类似ef -2的GTPase Efl1。为了检查这种释放是否涉及Efl1介导的rRNA结构重排，我们分析了三种情况下80s核糖体GTPase中心的rRNA构象：野生型、Delta Efl1和Delta Efl1的显性抑制因子R1。该分析仅限于25 S rRNA的结构域11和VI。R1核糖体的rRNA分析使我们能够区分Efl1缺失和Tif6核仁缺失的影响。Efl1抑制EF-2 GTPase活性，表明这两种蛋白具有相似的核糖体结合位点。两种类型的80s核糖体在分析的两个rRNA结构域中没有显示任何构象差异。然而，对自由60s亚基进行的相同分析显示，野生型和Delta efl1亚基之间存在一些rRNA构象差异，而来自抑制菌株的rRNA构象与野生型相似。这表明，在60 S预核糖体组装过程中，Tif6的核仁缺陷是Delta efl1 60 S亚基中观察到的rRNA构象变化的原因。我们还通过tap标记Tif6从三个遗传背景中纯化了60个S预核糖体。Delta efl1菌株中与Tif6p相关的60 S预糖体的蛋白质含量以较低的产量获得，但令人惊讶的是，其蛋白质组成与野生型和抑制菌株先验相似。(c) 2005 Elsevier Ltd版权所有。
Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Delta efl1 and a dominant suppressor R1 of Delta efl1. This analysis was restricted to domain 11 and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Delta efl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Delta efl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Delta efl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wildtype and the suppressor strain. (c) 2005 Elsevier Ltd. All rights reserved.