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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Construction of a fully active Cys-less elongation factor Tu: functional role of conserved cysteine 81.
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Construction of a fully active Cys-less elongation factor Tu: functional role of conserved cysteine 81.

机译：构建完全活性的Cys-less延伸因子Tu：保守的半胱氨酸81的功能作用。

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摘要

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.
机译：为了研究有效和准确的核糖体依赖性蛋白合成的基本翻译GTPase延长因子（EF）Tu的结构和功能要求，构建了无半胱氨酸（无Cys）突变体，可进行定点导入荧光和非荧光标记的重要性非常重要。然而，先前的报道表明，来自大肠杆菌的EF-Tu的81位的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无Cys的EF-Tu，我们分析了该位置上的125个细菌序列的序列变异，发现在少量序列中可以找到丙氨酸和蛋氨酸。在这里，我们报告了包含这些取代以及等位氨基酸丝氨酸的EF-Tu的三个少半胱氨酸变体的详细比较生化分析。通过表征核苷酸结合，EF-Ts相互作用，氨酰基-tRNA结合以及向核糖体的传递，我们证明在该位置只能耐受丙氨酸（或半胱氨酸），丝氨酸和蛋氨酸的取代显着削弱了氨酰基-tRNA，但是没有核苷酸结合。我们的发现表明EF-Tu 81位氨基酸残基相对于氨酰基-tRNA结合的关键功能。基于结构上的考虑，我们建议位置81通过螺旋B的精确定位间接有助于氨酰基-tRNA的结合。

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