Vlassov V V, Zuber G, Felden B, Behr J P, Giegé R
Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk.
Nucleic Acids Res. 1995 Aug 25;23(16):3161-7. doi: 10.1093/nar/23.16.3161.
Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjugates has been investigated. The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic virus RNA representing a minihelix capable of being enzymatically aminoacylated with histidine. Imidazole buffer and spermine-imidazole conjugates in the presence of free imidazole cleave phosphodiester bonds in the folded RNAs in a specific fashion. Imidazole buffer induces cleavages preferentially in single-stranded regions because nucleotides in these regions have more conformational freedom and can assume more easily the geometry needed for formation of the hydrolysis intermediate state. Spermine-imidazole constructs supplemented with free imidazole cleave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences. Hydrolysis patterns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA. Cleavages in a viral RNA fragment recapitulating a tRNA-like domain were found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imidazole-derived reagents as structural probes for solution mapping of RNAs. The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single- and double-stranded regions in RNAs.
对RNA在咪唑缓冲液中以及在精胺-咪唑缀合物作用下的水解情况进行了研究。RNA模型为酵母天冬氨酸转运RNA（tRNA(Asp)）以及一个源自烟草花叶病毒RNA 3'-末端序列的转录本，该转录本代表一种能够被组氨酸酶促氨酰化的小螺旋结构。在游离咪唑存在的情况下，咪唑缓冲液和精胺-咪唑缀合物以特定方式切割折叠RNA中的磷酸二酯键。咪唑缓冲液优先在单链区域诱导切割，因为这些区域的核苷酸具有更大的构象自由度，并且能够更轻松地呈现形成水解中间状态所需的几何形状。补充有游离咪唑的精胺-咪唑构建体在嘧啶残基后的单链区域内切割tRNA(Asp)，对嘧啶-A序列有明显偏好。水解模式表明，切割机制涉及静电结合的精胺-咪唑的咪唑残基以及游离咪唑在RNA最易接近的单链区域的攻击。在一个模拟tRNA样结构域的病毒RNA片段中发现的切割情况与该分子的模型一致，该模型解释了其功能特性，从而说明了咪唑衍生试剂作为RNA溶液图谱结构探针的潜力。切割反应操作简单，能提供反映RNA核糖磷酸骨架状态的信息，可用于绘制RNA中的单链和双链区域。
