Romby P, Giegé R, Houssier C, Grosjean H
J Mol Biol. 1985 Jul 5;184(1):107-118. doi: 10.1016/0022-2836(85)90047-6.
The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.
采用温度跃升法测量酵母天冬氨酸tRNA（反密码子GUC）双链体以及酵母天冬氨酸tRNA（GUC）-大肠杆菌缬氨酸tRNA（GAC）复合物的热力学和动力学参数。酵母天冬氨酸tRNA双链体在准自我互补反密码子的中间位置存在U/U错配，而在酵母天冬氨酸tRNA（GUC）-大肠杆菌缬氨酸tRNA（GAC）复合物中，tRNA具有互补反密码子。晶体结构中所证明的涉及GUC-GUC相互作用的天冬氨酸tRNA双链体的存在现已在溶液中得到证实。然而，与天冬氨酸tRNA和缬氨酸tRNA之间的复合物相比（0℃时缔合常数Kass = 4×10⁶ M⁻¹），其缔合常数的值（0℃时Kass = 10⁴ M⁻¹）表明该复合物相当弱，这种效应主要与解离速率常数的差异有关。经T1核糖核酸酶切割反密码子后的天冬氨酸tRNA未给出弛豫信号，这表明完整tRNA所观察到的效应完全归因于反密码子相互作用。对于其他具有准自我互补GNC反密码子（其中N为C、A或G）的tRNA，如大肠杆菌甘氨酸tRNA（GCC）、大肠杆菌缬氨酸tRNA（GAC）或大肠杆菌丙氨酸tRNA（GGC），未观察到双链体形成。这与如下观点相符：可能如同在晶体结构中一样，溶液中两个GUC反密码子之间形成了短双螺旋。由于空间效应，如果胞嘧啶、腺嘌呤或鸟嘌呤残基位于反密码子的中间位置，这种复合物的形成将会受到阻碍。在反密码子第一位具有修饰G残基（Q碱基）的大肠杆菌天冬氨酸tRNA，其自我缔合比酵母天冬氨酸tRNA弱，但其与大肠杆菌缬氨酸tRNA形成的复合物被发现仅比酵母天冬氨酸tRNA和大肠杆菌缬氨酸tRNA之间的复合物稳定性低1.5倍。在模拟用于酵母天冬氨酸tRNA结晶的条件（存在1.6 M硫酸铵和3 mM精胺）下进行的温度跃升实验表明，酵母和大肠杆菌天冬氨酸tRNA双链体或它们与大肠杆菌缬氨酸tRNA形成的复合物有显著稳定作用。该效应完全归因于硫酸铵；它是由熵驱动的，其影响反映在缔合速率常数上；未观察到对解离速率常数有影响。对于所有tRNA-tRNA复合物，加入硫酸铵后的解链温度显著升高。这项研究使得能够确定这样的溶液条件，即在其中具有合适反密码子的tRNA主要以反密码子-反密码子二聚体形式存在。
