Construction of a fully active Cys-less elongation factor Tu: Functional role of conserved cysteine 81
Construction of a fully active Cys-less elongation factor Tu: Functional role of conserved cysteine 81
复制标题
DOI：
10.1016/j.bbapap.2011.02.007
复制
发表时间：
2011-05-01
影响因子：
3.2
通讯作者：
Wieden, Hans-Joachim
中科院分区：
文献类型：
--
作者：
De Laurentiis, Evelina Ines;Mo, Fan;Wieden, Hans-Joachim
为了研究GTPase延伸因子（EF） Tu的结构和功能要求，高效准确地合成核糖体依赖蛋白，构建无半胱氨酸（Cys-less）突变体变体，允许定向引入荧光和非荧光标记是非常重要的。然而，先前的报道表明，大肠杆菌EF-Tu的81位半胱氨酸残基对其功能至关重要。为了研究半胱氨酸81的功能作用并构建一个完全活性的Cys-less EF-Tu，我们分析了125个细菌序列在该位置的序列变化，发现在少数序列中可以找到丙氨酸和蛋氨酸。在这里，我们报告了详细的比较生化分析含有这些取代以及等同氨基酸丝氨酸的三种不含cys的EF-Tu变体。通过对核苷酸结合、EF-Ts相互作用、氨基酰基- trna结合和核糖体递送的表征，我们证明只有丙氨酸（或半胱氨酸）可以在这个位置耐受，丝氨酸和蛋氨酸的取代会显著损害氨基酰基- trna，但不会损害核苷酸的结合。我们的研究结果表明，EF-Tu的81位氨基酸残基在氨基酰基- trna结合方面具有关键的功能作用。基于结构考虑，我们认为81号位置通过螺旋b的精确定位间接促进了氨基酰基- trna的结合。(C) 2011 Elsevier B.V.。
In order to study the structural and functional requirements of the essential translational GTPase elongation factor (EF) Tu for efficient and accurate ribosome-dependent protein synthesis, construction of a cysteine-free (Cys-less) mutant variant allowing for the site-directed introduction of fluorescent and non-fluorescent labels is of great importance. However, previous reports suggest that a cysteine residue in position 81 of EF-Tu from Escherichia coli is essential for its function. To study the functional role of cysteine 81 and to construct a fully active Cys-less EF-Tu, we have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitutions as well as the isosteric amino acid serine. By characterizing nucleotide binding, EF-Ts interaction, aminoacyl-tRNA binding, and delivery to the ribosome, we demonstrate that only alanine (or cysteine) can be tolerated in this position and that the serine and methionine substitutions significantly impair aminoacyl-tRNA, but not nucleotide binding. Our findings suggest a critical functional role of the amino acid residue in position 81 of EF-Tu with respect to aminoacyl-tRNA binding. Based on structural considerations, we suggest that position 81 indirectly contributes to aminoacyl-tRNA binding through the accurate positioning of helix B. (C) 2011 Elsevier B.V. All rights reserved.