Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu
Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu
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DOI：
10.1016/j.bbapap.2004.02.015
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发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Spremulli, LL
中科院分区：
文献类型：
--
作者：
Hunter, SE;Spremulli, LL
在蛋白质生物合成过程中，延伸因子 Tu (EF-Tu) 将氨酰基-tRNA (aa-tRNA) 递送至核糖体的 A 位点。这个因素在整个进化过程中高度保守。然而，细菌和哺乳动物线粒体 EF-Tu (EF-Tu(mt)) 的几个关键残基有所不同。此类残基之一是 Ser221（大肠杆菌编号）。该残基在细菌因子中作为 Ser 或 Thr 保守，但在 EF-Tu(mt) 中作为 Pro269 存在。与大肠杆菌 EF-Tu 相比，Pro269 重新定位包含该残基的环，并移动 EF-Tu(mt) 中相邻的 β 链，从而可能改变 aa-tRNA 受体茎的结合袋。 Pro269 在 EF-Tu(mt) 中突变为丝氨酸残基 (P269S)。为了进行比较，在大肠杆菌 EF-Tu (S221P) 中的 Ser221 处创建了互补突变。大肠杆菌 EF-Tu S221P 变体在大肠杆菌中表达较差，并且大多数分子无法折叠成活性构象。相反，EF-Tu(mt) P269S 在大肠杆菌中表达水平较高。当校正活性分子的百分比时，两种变体在使用大肠杆菌 Phe-tRNA(Phe) 和 Cys-tRNA(Cys) 形成三元复合物中与其各自的野生型因子一样有效。它们在 A 位点结合和大肠杆菌 Phe-tRNAPhe 体外翻译测定中也很活跃。此外，两种变体在三元复合物形成、A 位点结合和使用线粒体 Phe-tRNA(Phe) 的体外翻译测定中与其各自的野生型因子一样活跃。 (C) 2004 Elsevier B.V. 保留所有权利。
During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNAPhe. In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe). (C) 2004 Elsevier B.V. All rights reserved.