Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase
Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase
复制标题
DOI：
10.1128/jb.184.10.2692-2698.2002
复制
发表时间：
2002-05-01
影响因子：
3.2
通讯作者：
Bardwell, JCA
中科院分区：
文献类型：
--
作者：
Tan, J;Jakob, U;Bardwell, JCA
大肠杆菌RrmJ(FtsJ)热休克蛋白作为一种rRNA甲基转移酶，在完整的50S核糖体亚基中修饰23S rRNA的U2552位。RrmJ(FtsJ)基因的框内缺失在所有温度下都会导致严重的生长劣势，并导致核糖体亚基的大量积累，代价是功能上的70秒核糖体。为了研究其他大肠杆菌基因的过表达是否能够修复rrmJ缺失突变株的严重生长缺陷，我们从rrmJ缺失菌株构建了一个过表达文库，并克隆和鉴定了能够挽救rrmJ突变表型的大肠杆菌基因。我们的目的是确定其他甲基酶，它们的特异性与RrmJ的特异性足够重叠，以便在过度表达时进行互补。令我们惊讶的是，这种方法没有发现甲基酶；相反，当两个小的GTP酶Obg(YhbZ)和EngA在rrmJ缺失株中过表达时，被发现恢复了原本严重受损的核糖体组装过程和/或7S核糖体的稳定性。从这些高表达菌株制备的50S核糖体亚基仍然可以作为纯化的RrmJ的体外底物，表明23S rRNA可能仍然缺乏高度保守的Um2552修饰。然而，这种修饰的明显缺失不再导致核糖体缺陷或生长劣势。另一种相关的小GTP酶Era的大量过表达未能挽救rrmJ菌株的生长缺陷。这些发现表明rRNA甲基化和GTP酶功能之间存在迄今意想不到的联系，特别是两个小的GTP酶Obg和EngA的联系。
The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.