Amano M, Kyogoku Y
Institute for Protein Research, Osaka University, Japan.
Eur J Biochem. 1993 Oct 1;217(1):131-6. doi: 10.1111/j.1432-1033.1993.tb18227.x.
NMR spectra of Bombyx mori tRNA(GCCGly) were recorded in the GCC absence and presence of the oligonucleotide, GGCUp, which contains the codon sequence, GGC. The difference between the spectra with and without the codon oligonucleotide indicates the appearance of five new imino proton peaks. For the assignment of these peaks, GGCUp, in which the 5'-terminal G was enriched with 95% 15N, was prepared (G, 15N-labeled guanosine). In the imino proton spectrum of B. mori tRNA(GCCGly) on the addition of G*GCUp, the peak at 12 ppm became a doublet due to coupling with 15N nuclei. In the two-dimensional 1H-15N heteronuclear multiple-quantum correlation (HMQC) spectrum, only the peak at 12 ppm was observed, and thus it was assigned to the imino proton of the 5'-terminal G of GGCUp interacting with tRNA(GCCGly). Judging from the temperature effect and chemical shifts, the five new imino proton peaks are presumed to be due to three G.C base pairs, induced by the codon-anticodon interaction, and one U.U base pair, induced by an interaction between the 3' terminal U of GGCUp and U33 neighboring the anticodon. The binding of three trinucleotides (GGCp, GGUp and GCUp) to B. mori tRNA(GCCGly) was also investigated. Ultracentrifugation analysis showed that tRNA(GCCGly) underwent dimerization through the anticodon-anticodon interaction, but the dimerization was broken on addition of GGCUp. On 1H-NMR and ultracentrifugation analysis, it was found that GCUp not complementary to the anticodon also binds to the anticodon loop.
在寡核苷酸GGCUp（其包含密码子序列GGC）不存在和存在的情况下，记录了家蚕tRNA（GCCGly）的核磁共振谱。有密码子寡核苷酸和无密码子寡核苷酸时的光谱差异表明出现了五个新的亚氨基质子峰。为了对这些峰进行归属，制备了5'-末端G富含95% 15N的GGCUp（G，15N标记的鸟苷）。在添加G*GCUp后家蚕tRNA（GCCGly）的亚氨基质子谱中，12 ppm处的峰由于与15N核的偶合而变成了双峰。在二维1H-15N异核多量子相关（HMQC）谱中，仅观察到12 ppm处的峰，因此将其归属为与tRNA（GCCGly）相互作用的GGCUp 5'-末端G的亚氨基质子。从温度效应和化学位移判断，这五个新的亚氨基质子峰推测是由密码子-反密码子相互作用诱导的三个G·C碱基对以及GGCUp 3'-末端U与反密码子相邻的U33之间的相互作用诱导的一个U·U碱基对引起的。还研究了三种三核苷酸（GGCp、GGUp和GCUp）与家蚕tRNA（GCCGly）的结合。超速离心分析表明，tRNA（GCCGly）通过反密码子-反密码子相互作用发生二聚化，但添加GGCUp后二聚化被打破。通过1H-NMR和超速离心分析发现，与反密码子不互补的GCUp也与反密码子环结合。
