Gabryszuk J, Przykorska A, Monko M, Kuligowska E, Sturchler C, Krol A, Dirheimer G, Szarkowski J W, Keith G
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Gene. 1995 Aug 19;161(2):259-63. doi: 10.1016/0378-1119(95)00287-g.
Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA(Sec). To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA(Sec) gene. We found that the structures in solution of the native tRNA(Sec) and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA(Sec), containing 5-methylcarboxymethyluridine (mcm5U34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A36 and A38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i6A37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA(Sec) than in the transcript.
在植物中发现的两种单链特异性核酸酶已被用于研究天然牛肝硒代半胱氨酸tRNA（Sec）的二级和三级结构。为了检查核苷酸修饰对这些结构的可能影响，我们将用完全修饰的tRNA得到的结果与通过非洲爪蟾tRNA（Sec）基因的体外T7转录制备的未修饰转录本的结果进行了比较。我们发现，尽管在对酶探针的可及性方面存在一些差异，但天然tRNA（Sec）和转录本在溶液中的结构非常相似。实际上，天然牛tRNA（Sec）的修饰反密码子环，含有5-甲基羧甲基尿苷（mcm5U34）和N6-异戊烯基腺苷（i6A37），与转录本相比，对Rn核酸酶的可及性较低：与转录本相比，代表A36和A38处切割的条带强度要低得多，而在天然分子的反密码子环中i6A37水平处未发现切割。令人惊讶的是，已发现天然分子的可变臂更容易受到单链特异性核酸酶的作用，这表明天然牛tRNA（Sec）中可变臂的结构比转录本中的更松散。
