Douthwaite S, Garrett R A, Wagner R
Eur J Biochem. 1983 Mar 15;131(2):261-9. doi: 10.1111/j.1432-1033.1983.tb07258.x.
tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF-Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A73-C-C-A sequence of the aminoacyl stem, the anticodon loop, the D-loop and the extra loop and the results correlate well with the three-dimensional structure of tRNAPheyeast determined by X-ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3' terminus with pCp. In the ternary complex, with elongation factor EF-Tu and GTP, changes in modification were observed at two sites, A73-C-C-A at the 3' terminus and C-13 and C-17 in the D-loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNAPhe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C-74 and C-75 were strongly protected at both sites, the adjacent A-73 showed an enhanced reactivity in the A site. The anticodon region G34-A-A-ms2.6(1)A was also strongly protected at both sites. In addition, nucleotide A-21 was protected during A-site, but not P-site, binding.
使用Peattie和Gilbert [《美国国家科学院院刊》，77, 4679 - 4689 (1980)] 所描述的化学修饰方法，对大肠杆菌苯丙氨酸转运核糖核酸（tRNAPheE.coli）中可及的鸟苷、胞苷和腺苷残基进行修饰。比较了处于游离状态、与延伸因子EF - Tu和GTP形成的三元复合物状态以及核糖体A和P位点状态下tRNA的修饰特征。设计了一种特殊程序，专门监测氨酰化状态的tRNA分子。在游离tRNA中，反应性最强的碱基局限于氨酰基茎的A73 - C - C - A序列、反密码子环、D环和额外环，其结果与通过X射线研究确定的酵母苯丙氨酸tRNA的三维结构密切相关。无论用苯丙氨酸对tRNA进行氨酰化，还是用对氯苯磷二酚（pCp）标记3'末端，反应性模式均未受影响。在与延伸因子EF - Tu和GTP形成的三元复合物中，在两个位点观察到修饰变化，一个是3'末端的A73 - C - C - A，另一个是D环区域的C - 13和C - 17，这两个位点相距约6纳米；在反密码子环中未观察到差异。结合在核糖体A或P位点的tRNAPhe表现出相似但不完全相同的修饰模式。虽然核苷酸C - 74和C - 75在两个位点均受到强烈保护，但相邻的A - 73在A位点表现出增强的反应性。反密码子区域G34 - A - A - ms2.6(1)A在两个位点也受到强烈保护。此外，核苷酸A - 21在A位点结合期间受到保护，但在P位点结合时未受保护。
