Nagamatsu K, Miyazawa Y
J Biochem. 1983 Dec;94(6):1967-71. doi: 10.1093/oxfordjournals.jbchem.a134550.
The A76 or A73 nucleotide at the 3'end of tRNAPhe was modified with the fluorescent reagent of proflavine (PF). The distance between the fluorophore of the 3'end and the Y base was measured by singlet-singlet energy transfer under the conditions of 10 mM and 0.01 mM Mg2+. The distance obtained at 10 mM Mg2+ is very close to that obtained by the X-ray diffraction method, while the distance at 0.01 mM Mg2+ is significantly smaller. The difference in the distance is explained as a result of destabilization of the tertiary structure with reduction of the Mg2+ concentration. The calculated distance between A73 and A76 shows the stacked conformation of the CCA strand. Fluorescent quenching experiments showed that the degree of stabilization of 3'end A76 by stacking is lower than that of A73. The removal of the CCA segment causes a difference not only in the thermal melting curves but also in the fluorescent wavelength of the Y base at 0.01 mM Mg2+. The results suggest that the 3'end CCA strand has a helical structure and contributes to the stabilization of the whole structure of tRNAPhe.
用荧光试剂硫酸原黄素（PF）对苯丙氨酸转运RNA（tRNAPhe）3'端的A76或A73核苷酸进行修饰。在10 mM和0.01 mM Mg2+条件下，通过单重态-单重态能量转移测量3'端荧光团与Y碱基之间的距离。在10 mM Mg2+条件下获得的距离与通过X射线衍射法获得的距离非常接近，而在0.01 mM Mg2+条件下的距离明显更小。距离的差异被解释为随着Mg2+浓度降低，三级结构不稳定的结果。计算得到的A73和A76之间的距离显示了CCA链的堆积构象。荧光猝灭实验表明，通过堆积对3'端A76的稳定程度低于A73。去除CCA片段不仅会导致热熔解曲线不同，还会导致在0.01 mM Mg2+条件下Y碱基的荧光波长不同。结果表明，3'端CCA链具有螺旋结构，并有助于稳定tRNAPhe的整体结构。
