Nureki O, Suzuki K, Hara-Yokoyama M, Kohno T, Matsuzawa H, Ohta T, Shimizu T, Morikawa K, Miyazawa T, Yokoyama S
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Eur J Biochem. 1992 Mar 1;204(2):465-72. doi: 10.1111/j.1432-1033.1992.tb16656.x.
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.
利用编码谷氨酰胺 - tRNA合成酶N端氨基酸序列的合成寡核苷酸探针，从嗜热栖热菌HB8（Thermus thermophilus HB8）中分离出谷氨酰胺 - tRNA合成酶基因。核苷酸序列分析揭示了一个编码由468个氨基酸残基组成的蛋白质的开放阅读框（Mr 53,901）。嗜热栖热菌谷氨酰胺 - tRNA合成酶基因的密码子使用情况实际上与栖热菌属细菌蛋白质基因的特征使用情况相似：密码子第三位的G + C含量高达94%。相比之下，嗜热栖热菌谷氨酰胺 - tRNA合成酶的氨基酸序列与细菌谷氨酰胺 - tRNA合成酶具有高度相似性（同一性为35 - 45%）；ATP和tRNA（Glu）3'末端结合位点的序列高度保守。谷氨酰胺 - tRNA合成酶基因在tac启动子的控制下在大肠杆菌中高效表达。重组嗜热栖热菌谷氨酰胺 - tRNA合成酶具有极高的热稳定性，通过热处理和三步柱层析纯化至均一性。通过气相扩散技术从聚乙二醇6000溶液中获得了嗜热栖热菌谷氨酰胺 - tRNA合成酶的单晶。这些晶体对X射线的衍射分辨率超过0.35 nm。该晶体属于正交空间群P2(1)2(1)2(1)，晶胞参数为a = 8.64 nm，b = 8.86 nm，c = 8.49 nm。
