Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine

FEBS Letters

Sprinzl, Mathias¹  Reza Ahmadian, M.¹  Kreutzer, Roland¹  Blechschmidt, Bernd¹ 

[1] Laboratorium für Biochemie Universität Bayreuth, 95440 Bayreuth, Germany

关键词: Elongation factor Tu;    GTPase;    GDP/GTP-binding protein;    DTT;    dithiothreitol;    EF-Ts;    elongation factor Ts;    EF-Tu;    elongation factor Tu;    GAP;    GTPase activating protein;    MOPS;    3-(N-morpholino)propanesulfonic acid;    [IASNE-s2C75]Tyr-tRNATyr;    Tyr-tRNATyr alkylated with N-iodacetyl-N′-(5-sulfo-1-napthyl)-ethylendiamin on the 2-thiocytidine incorporated into position 75;   

DOI  :  10.1016/0014-5793(95)01354-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A·GTP complex is almost 40 times lower as compared to the native EF-Tu·GTP. These observations are in agreement with the tertiary structure of EF-Tu·GTP, in which threonine-62 is interacting with the Mg²⁺ ion, γ-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

【 授权许可】

Unknown