Gornicki P, Nurse K, Hellmann W, Boublik M, Ofengand J
J Biol Chem. 1984 Aug 25;259(16):10493-8.
A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E. coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA. By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy. The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid. When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked. Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification. The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented. Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion. Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site.
以往的一系列研究表明，当将大肠杆菌tRNAVal1置于大肠杆菌核糖体的P位点并进行辐照时，该tRNA的5'-反密码子碱基，即5-羧甲氧基尿苷，会与16 S rRNA的C-1400发生交联。通过用一个通过9 Å间隔连接的2,4-二硝基苯基（DNP）基团标记交联tRNA残基的羧基，就有可能通过免疫电子显微镜直接观察到这个交联位点。通过向羧基添加乙二胺来连接DNP基团，随后使新形成的游离氨基与N-2,4-二硝基苯基-γ-氨基丁酸的N-羟基琥珀酰亚胺酯缩合。当与抗DNP抗体反应时，这种修饰会使抗体表面靠近交联的嘧啶环9 Å以内。tRNA的修饰对密码子依赖性结合和交联均无实质性影响。只有当50-30 S亚基的结合被阻止时，tRNA-核糖体加合物才会与抗DNP抗体形成稳定的复合物。对免疫复合物的电子显微镜检查表明，检测到的复合物中超过95%的抗体位于将30 S亚基的头部和颈部与大突起分隔开的裂隙深处。由于这是tRNA反密码子交联的位点，我们得出结论，30 S亚基上的这个区域对应于解码位点。
