Michałowski D, Wrzesinski J, Krzyzosiak W
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
Biochemistry. 1996 Aug 20;35(33):10727-34. doi: 10.1021/bi9530393.
The U59 and C60 residues, which form the strong Pb(II) ion binding site in yeast tRNA(Phe), were systematically mutated, and the effects of individual mutations on cleavages induced by various metal ions were analyzed. It turned out that the presence of C60 is essential for efficient cleavage of the D-loop by Pb(II), Eu(III), and Mg(II)ions. On the other hand, manganese ions were capable of cleaving these mutants with an efficiency similar to that observed for the wild type transcript. Moreover, in all C60 mutants, the main Mn(II) cleavage site was shifted by one phosphate from P16 to P17. Mutations of U59 did not affect so dramatically the efficiency and specificity of the D-loop hydrolysis induced by all studied metal ions. In the G59C60 mutant cleaved by Pb(II) ions, new cuts took place in the T-stem at P63-P65. Also, the C60 mutants were cleaved more strongly at P49 by Pb(II) ions. In G59C60 and A59C60 as well as in all C60 mutants, the Mg(II) and Mn(II) cleavage at P61 was suppressed. Nevertheless, the changes in overall tRNA structure resulting from U59 and C60 mutations were rather subtle. The studied mutants showed S1 and V1 nuclease digestion patterns practically indistinguishable from those observed in the wild type transcript. The metal ions are shown to be well-suited for monitoring the local changes in the structure of the investigated tRNA variants and when used as a set of probes can give a more complete picture of changes that occur in transcripts as a result of a mutation.
在酵母苯丙氨酸转运RNA（tRNA(Phe)）中形成强铅离子（Pb(II)）结合位点的U59和C60残基被系统地突变，并分析了单个突变对各种金属离子诱导切割的影响。结果表明，C60的存在对于Pb(II)、铕离子（Eu(III)）和镁离子（Mg(II)）有效切割D环至关重要。另一方面，锰离子能够以与野生型转录本相似的效率切割这些突变体。此外，在所有C60突变体中，主要的锰离子（Mn(II)）切割位点从P16的一个磷酸处移至P17。U59的突变对所有研究金属离子诱导的D环水解效率和特异性没有如此显著的影响。在被Pb(II)离子切割的G59C60突变体中，在T茎的P63 - P65处出现了新的切割。同样，C60突变体在P49处被Pb(II)离子更强烈地切割。在G59C60和A59C60以及所有C60突变体中，P61处的Mg(II)和Mn(II)切割受到抑制。然而，由U59和C60突变导致的tRNA整体结构变化相当细微。所研究的突变体显示出的S1和V1核酸酶消化模式与野生型转录本中观察到的模式几乎无法区分。金属离子被证明非常适合监测所研究的tRNA变体结构的局部变化，并且当作为一组探针使用时，可以更全面地呈现由于突变而在转录本中发生的变化情况。
