Identification of peptide sequences at the tRNA binding site of Escherichia coli methionyl-tRNA synthetase.
Identification of peptide sequences at the tRNA binding site of Escherichia coli methionyl-tRNA synthetase.
复制标题
大肠杆菌甲硫氨酰-tRNA 合成酶 tRNA 结合位点肽序列的鉴定。
DOI：
10.1021/bi00364a015
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Schulman,LH
中科院分区：
文献类型：
--
作者：
Valenzuela,D;Schulman,LH
修订稿于 1986 年 4 月 7 日收到摘要：通过使用携带赖氨酸反应性交联剂的 tRNA 衍生物，将大肠杆菌 tRNA™ 61 的四个不同结构区域共价偶联至大肠杆菌甲硫氨酰-tRNA 合成酶 (MetRS)。我们之前已经表明，这种交联发生在酶的 tRNA 结合位点，并且仅涉及蛋白质中少量潜在可用的赖氨酸残基的反应 [Schulman, L. H., Valenzuela, D., & Pelka, H.(1981) Biochemistry 20, 6018-6023； Valenzuela, D.、Leon, O. 和 Schulman, LH (1984) 生物化学。生物物理学。资源。交流。 119、677-684]。在这项工作中，已鉴定出四种交联肽。用胰蛋白酶消化 tRNA-蛋白质交联复合物，并通过阴离子交换色谱将附着在 tRNA 上的肽与大部分胰蛋白酶肽分离。 tRNA 结合的肽通过交联剂二硫键的裂解而释放，并通过反相高压液相色谱分离，产生五个主峰。氨基酸分析表明其中四个峰含有单个肽。序列分析表明，这些肽通过 MetRS 一级序列中的赖氨酸残基 402、439、465 和 640 与 tRNA™ 61 交联。因此，tRNA 的结合涉及与 MetRS 羧基末端一半的相互作用，而 X 射线晶体学数据显示 ATP 结合位点位于蛋白质的 N 末端结构域 [Zelwer/C, Risler, J. L., & Brunie, S.(1982) J. Mol.生物。 155, 63-81], 本研究开发的方法应适用于测定其他蛋白质 tRNA 结合位点的肽序列。
Revised Manuscript Received April 7, 1986 abstract: Four different structural regions of Escherichia coli tRNA™ 61have been covalently coupled to E. coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker. We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysineresidues in the protein [Schulman, L. H., Valenzuela, D., & Pelka, H.(1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, LH (1984) Biochem. Biophys. Res. Commun. 119, 677-684]. In this work, four of the cross-linked peptides have been identified. The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography. The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks. Amino acid analysis indicated that four of these peaks contained single peptides. Sequence analysis showed that thepeptides were cross-linked to tRNA™ 61 through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS. Binding of the tRNA therefore involves interactionswith the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein [Zelwer/C, Risler, J. L., & Brunie, S.(1982) J. Mol. Biol. 155, 63-81], The methods developed for the present studies should be applicable to determination of peptide sequences atthe tRNA binding sites of other proteins.