Karring Henrik, Björnsson Asgeir, Thirup Søren, Clark Brian F C, Knudsen Charlotte R
Department of Molecular Biology, Aarhus University, Denmark.
Eur J Biochem. 2003 Nov;270(21):4294-305. doi: 10.1046/j.1432-1033.2003.03822.x.
Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.
延伸因子Ts（EF-Ts）是延伸因子Tu（EF-Tu）的鸟嘌呤核苷酸交换因子，负责促进氨酰tRNA与mRNA编程核糖体的结合。大肠杆菌EF-Tu-EF-Ts复合物的结构揭示了EF-Ts中一个突出的反平行卷曲螺旋基序，它负责晶体中EF-Ts的二聚化。在本研究中，通过基因替换从大肠杆菌基因组中删除了编码EF-Ts中卷曲螺旋基序的序列。根据所使用的生长条件，所得突变菌株的生长速率为野生型菌株的70-95%。突变菌株能感知氨基酸饥饿，并在比野生型菌株更低的细胞密度下合成鸟苷5'-二磷酸3'-二磷酸和鸟苷5'-三磷酸3'-二磷酸。卷曲螺旋基序的缺失仅部分降低了EF-Ts刺激EF-Tu中鸟嘌呤核苷酸交换的能力。然而，解离突变型EF-Tu-EF-Ts复合物所需的鸟嘌呤核苷酸（GDP和GTP）浓度比野生型复合物至少低两个数量级。结果表明，卷曲螺旋基序在EF-Ts与鸟嘌呤核苷酸竞争结合EF-Tu的能力中起重要作用。目前的结果还表明，该缺失改变了EF-Ts和奇霉素竞争结合EF-Tu的情况。
