Hayashi I, Kawai G, Watanabe K
Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Tokyo, Bunkyo-ku, 113-8656, Japan.
J Mol Biol. 1998 Nov 20;284(1):57-69. doi: 10.1006/jmbi.1998.2151.
Although mammalian mitochondrial serine-specific tRNA with the anticodon UGA (tRNASerUGA) appears to possess an almost normal cloverleaf secondary structure, it exhibits an extraordinarily low melting temperature (tm). An in vitro tRNASerUGA transcript without modified nucleosides had an even lower tm and slightly less hyperchromicity, but its tertiary structure was apparently very similar to that of the native counterpart judging from its aminoacylation activity and the body of experimental evidence so far obtained for canonical tRNAs. The transcript was therefore used to investigate the higher-order structure and thermal instability of tRNASerUGA. 1H-NMR analysis of the transcript showed that it takes a nearly L-shaped tertiary structure with similar tertiary base-pairings to those found in yeast tRNAPhe, which is representative of canonical tRNAs. However, magnesium ion titration revealed that Mg2+ affected the chemical shifts of the tRNASerUGA transcript differently than those of canonical tRNAs so far studied; the former was less sensitive toward Mg2+, especially in the D-arm region. This observation was confirmed by NMR analysis with paramagnetic manganese ion titration. Hill plots derived from the CD spectral changes caused by titration with Mg2+ suggested that the tRNASerUGA transcript had fewer Mg2+ binding sites than those of yeast tRNAPhe as well as its transcript, a finding that was consistent with the NMR data. We thus surmise that the thermal instability of both the transcript and tRNASerUGA itself originated from a reduction in the number of the divalent ion binding sites within the tRNA molecule. These results suggest a new type of thermal instability for mitochondrial tRNA.
尽管带有反密码子UGA的哺乳动物线粒体丝氨酸特异性tRNA（tRNASerUGA）似乎具有几乎正常的三叶草二级结构，但其解链温度（tm）却异常低。一个没有修饰核苷的体外tRNASerUGA转录本具有更低的tm和稍低的增色效应，但其三级结构从其氨基酰化活性和目前针对典型tRNA所获得的实验证据来看，显然与天然对应物非常相似。因此，该转录本被用于研究tRNASerUGA的高级结构和热不稳定性。对该转录本的1H-NMR分析表明，它具有近乎L形的三级结构，其三级碱基配对与酵母tRNAPhe（典型tRNA的代表）中的相似。然而，镁离子滴定显示，Mg2+对tRNASerUGA转录本化学位移的影响与迄今研究的典型tRNA不同；前者对Mg2+不太敏感，尤其是在D臂区域。用顺磁性锰离子滴定的NMR分析证实了这一观察结果。由Mg2+滴定引起的CD光谱变化得出的希尔图表明，tRNASerUGA转录本的Mg2+结合位点比酵母tRNAPhe及其转录本的少，这一发现与NMR数据一致。因此我们推测，转录本和tRNASerUGA本身的热不稳定性都源于tRNA分子内二价离子结合位点数量的减少。这些结果表明线粒体tRNA存在一种新型的热不稳定性。
