来自大肠杆菌的核糖体蛋白S1的第二个OB折叠对TmRNA识别的贡献。

Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA.

作者信息

Okada Takahiro, Wower Iwona K, Wower Jacek, Zwieb Christian W, Kimura Makoto

机构信息

Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.

出版信息

Biosci Biotechnol Biochem. 2004 Nov;68(11):2319-25. doi: 10.1271/bbb.68.2319.

DOI:10.1271/bbb.68.2319

PMID:15564671

Abstract

Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds). In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes. The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA. Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity. Furthermore, the E. coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E. coli S1, showed no interaction with E. coli tmRNA, as judged by the results of a gel shift assay. Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)). These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.

摘要

大肠杆菌核糖体蛋白S1由六个重复的同源寡核苷酸/寡糖结合折叠结构域（OB折叠）组成。在反式翻译过程中，S1在将转运信使RNA（tmRNA）递送至停滞的核糖体中发挥作用。在tmRNA存在的情况下，发现S1的第二个OB折叠结构域可免受胰蛋白酶消化的影响。缺失了第一个和第二个OB折叠结构域的截短型S1突变体Delta2显示出tmRNA结合活性显著降低。此外，通过凝胶迁移试验结果判断，来自枯草芽孢杆菌的大肠杆菌S1同源物（BS1），其对应于大肠杆菌S1的四个C末端OB折叠结构域，与大肠杆菌tmRNA没有相互作用。表面等离子体共振分析表明，与野生型蛋白S1（ka，3.32×10³ M⁻¹·s⁻¹；kd，0.56×10⁻³ s⁻¹）相比，突变体Delta2和BS1的结合速率常数降低（ka，0.59×10³ M⁻¹·s⁻¹；和ka，1.89×10³ M⁻¹·s⁻¹），而它们各自的解离速率常数保持不变（kd，0.67×10⁻³ s⁻¹；和kd，0.53×10⁻³ s⁻¹）。这些结果表明，蛋白S1中的第二个OB折叠结构域对于tmRNA的识别至关重要，而四个C末端OB折叠结构域在稳定S1 - tmRNA复合物中发挥作用。