Romby P, Moras D, Dumas P, Ebel J P, Giegé R
Institut de Biologie Moléculaire et Cellulaire, Centre de le Recherche Scientifique, Strasbourg, France.
J Mol Biol. 1987 May 5;195(1):193-204. doi: 10.1016/0022-2836(87)90336-6.
A comparative study of the solution structures of yeast tRNA(Asp) and tRNA(Phe) was undertaken with chemical reagents as structural probes. The reactivity of N-7 positions in guanine and adenine residues was assayed with dimethylsulphate and diethyl-pyrocarbonate, respectively, and that of the N-3 position in cytosine residues with dimethylsulphate. Experiments involved statistical modifications of end-labelled tRNAs, followed by splitting at modified positions. The resulting end-labelled oligonucleotides were resolved on polyacrylamide sequencing gels and analysed by autoradiography. Three different experimental conditions were used to follow the progressive denaturation of the two tRNAs. Experiments were done in parallel on tRNA(Asp) and tRNA(Phe) to enable comparison between the two solution structures and to correlate the results with the crystalline conformations of both molecules. Structural differences were detected for G4, G45, G71 and A21: G4 and A21 are reactive in tRNA(Asp) and protected in tRNA(Phe), while G45 and G71 are protected in tRNA(Asp) and reactive in tRNA(Phe). For the N-7 atom of A21, the different reactivity is correlated with the variable variable loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46. For G4 and G71, the differential reactivities are linked to a different stacking in both tRNAs. This observation is of general significance for helical stems. If the previous results could be fully explained by the crystal structures, unexpected similarities in solution were found for N-3 alkylation of C56 in the T-loop, which according to crystallography should be reactive in tRNA(Asp). The apparent discrepancy is due to conformational differences between crystalline and solution tRNA(Asp) at the level of the D and T-loop contacts, linked to long-distance effects induced by the quasi-self-complementary anticodon GUC, which favour duplex formation within the crystal, contrarily to solution conditions where the tRNA is essentially in its free state.
以化学试剂作为结构探针，对酵母天冬氨酸转运核糖核酸（tRNAAsp）和苯丙氨酸转运核糖核酸（tRNAPhe）的溶液结构进行了比较研究。分别用硫酸二甲酯和焦碳酸二乙酯测定鸟嘌呤和腺嘌呤残基中N-7位的反应活性，用硫酸二甲酯测定胞嘧啶残基中N-3位的反应活性。实验包括对末端标记的转运核糖核酸进行统计学修饰，然后在修饰位点进行切割。将得到的末端标记寡核苷酸在聚丙烯酰胺测序凝胶上分离，并用放射自显影法进行分析。采用三种不同的实验条件来跟踪两种转运核糖核酸的逐步变性。对tRNAAsp和tRNAPhe同时进行实验，以便比较两种溶液结构，并将结果与两种分子的晶体构象相关联。检测到G4、G45、G71和A21存在结构差异：G4和A21在tRNAAsp中具有反应活性，在tRNAPhe中受到保护，而G45和G71在tRNAAsp中受到保护，在tRNAPhe中具有反应活性。对于A21的N-7原子，不同的反应活性与两种转运核糖核酸中可变环结构的差异有关；对于G45，结果可以用G45和46位残基之间A9的不同堆积来解释。对于G4和G71，不同的反应活性与两种转运核糖核酸中不同的堆积有关。这一观察结果对螺旋茎具有普遍意义。如果先前的结果可以用晶体结构完全解释，那么在溶液中发现了T环中C56的N-3烷基化存在意想不到的相似性，根据晶体学，这在tRNAAsp中应该具有反应活性。明显的差异是由于晶体态和溶液态tRNAAsp在D环和T环接触水平上的构象差异，这与准自我互补反密码子GUC诱导的长距离效应有关，准自我互补反密码子GUC有利于晶体中的双链形成，这与溶液条件相反，在溶液条件下转运核糖核酸基本上处于自由状态。
