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首页> 外文期刊>Biochemistry >Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N-2-methylguanosine
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Identifying RNA minor groove tertiary contacts by nucleotide analogue interference mapping with N-2-methylguanosine

机译：通过N-2-甲基鸟苷的核苷酸类似物干扰图谱识别RNA小沟三级接触

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摘要

Nucleotide analogue interference mapping (NAIM) is a general biochemical method that rapidly identifies the chemical groups important for RNA function. In principle, NAIM can be extended to any nucleotide that can be incorporated into an in vitro transcript by an RNA polymerase. Here we report the synthesis of 5'-O-(1-thio)-N-2-methylguanosine triphosphate (m(2)G alpha S) and its incorporation into two reverse splicing forms of the Tetrahymena group I intron using a mutant form of T7 RNA polymerase. This analogue replaces one proton of the N2 exocyclic amine with a methyl group, but is as stable as guanosine (G) for secondary structure formation. We have identified three sites of m(2)G alpha S interference within the Tetrahymena intron: G22, G212, and G303. All three of these guanosine residues are known to utilize their exocyclic amino groups to participate in tertiary hydrogen bonds within the ribozyme structure. Unlike the interference pattern with the phosphorothioate of inosine (I alpha S, an analogue that deletes the N2 amine of G), m(2)G alpha S substitution did not cause interference at positions attributable to secondary structural stability effects. Given that the RNA minor groove is likely to be widely used for helix packing, m(2)G alpha S provides an especially valuable reagent to identify RNA minor groove tertiary contacts in less well-characterized RNAs. [References: 54]
机译：核苷酸类似物干扰图谱（NAIM）是一种通用的生化方法，可快速识别出对RNA功能重要的化学基团。原则上，NAIM可以扩展到可以通过RNA聚合酶掺入体外转录物中的任何核苷酸。在这里我们报告5'-O-（1-硫）-N-2-甲基鸟苷三磷酸（m（2）G alpha S）的合成及其使用突变形式并入四膜虫第一类内含子的两个反向剪接形式T7 RNA聚合酶。该类似物用甲基取代一个N2环外胺的质子，但与鸟嘌呤（G）一样稳定，可形成二级结构。我们在四膜虫内含子中鉴定了m（2）G alpha S干扰的三个位点：G22，G212和G303。已知所有这三个鸟苷残基均利用其环外氨基来参与核酶结构内的叔氢键。与肌苷的硫代磷酸酯（I alpha S，一种删除G的N2胺的类似物）的干扰模式不同，m（2）G alpha S取代不会在可归因于二级结构稳定性的位置引起干扰。鉴于RNA小沟很可能被广泛用于螺旋堆积，m（2）G alpha S提供了一种特别有价值的试剂，可用于识别特征较少的RNA中的RNA小沟三级接触。 [参考：54]

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