Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping.
Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping.
复制标题
通过核苷酸类似物干扰图谱定义四膜虫 I 组内含子功能所必需的化学基团。
DOI：
10.1073/pnas.94.7.2903
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发表时间：
1997
影响因子：
11.1
通讯作者：
Shetty,K
中科院分区：
文献类型：
--
作者：
Strobel,SA;Shetty,K
需要改进的原子分辨率生化方法来识别 RNA 中对其活性至关重要的化学基团。作为实现这一目标的一步，我们报告了在核苷酸类似物干扰图谱 (NAIM) 测定中使用 5'-O-(1-硫代)肌苷单磷酸 (IMPαS)，这使得可以同时但单独地确定大 RNA 中几乎每个 G 的 N2 环外胺的贡献。使用 IMPαS，我们鉴定了四膜虫组 I 内含子 5' 或 3' 外显子连接所必需的环外胺。我们报道了三个系统发育上保守的鸟苷（G111、G112 和 G303）的氨基对于 3' 外显子连接很重要。 G22 的胺以及 P1 螺旋内其他四个鸟苷的胺对于 5' 外显子的连接至关重要。先前的研究表明，G22 或 G303 点突变为腺苷 (A) 会显着降低活性。与肌苷一样，腺苷缺乏 N2 氨基。使用 5'-O-(1-硫代)二氨基嘌呤核苷一磷酸 (DMPαS)（一种具有 N2 环外胺的腺苷类似物），通过 NAIM 在突变位点检测到 G22A 和 G303A 点突变的干扰拯救。 G22A 点突变体也可以通过在 A24 处掺入 DMPαS 来挽救。通过类比遗传学，存在类似于功能丧失、回复和抑制的干扰表型。该方法可以很容易地扩展到其他核苷酸类似物，用于分析各种 RNA 和 DNA 活性所必需的化学基团。
Improved atomic resolution biochemical methods are needed to identify the chemical groups within an RNA that are essential to its activity. As a step toward this goal, we report the use of 5′-O-(1-thio)inosine monophosphate (IMPαS) in a nucleotide analog interference mapping (NAIM) assay that makes it possible to simultaneously, yet individually, determine the contribution of almost every N2 exocyclic amine of G within a large RNA. Using IMPαS, we identified the exocyclic amines that are essential for 5′ or 3′ exon ligation by theTetrahymenagroup I intron. We report that the amino groups of three phylogenetically conserved guanosines (G111, G112, and G303) are important for 3′ exon ligation. The amine of G22, as well as the amines of the other four guanosines within the P1 helix, are essential for ligation of the 5′ exon. Previous work has shown that point mutation of either G22 or G303 to an adenosine (A) substantially reduces activity. Like inosine, adenosine lacks an N2 amino group. Interference rescue of the G22A and G303A point mutations was detected at the site of mutation by NAIM using 5′-O-(1-thio)diaminopurine riboside monophosphate (DMPαS), an adenosine analog that has an N2 exocyclic amine. The G22A point mutant could also be rescued by incorporation of DMPαS at A24. By analogy to genetics, there are interference phenotypes comparable to loss of function, reversion, and suppression. This method can be readily extended to other nucleotide analogs for the analysis of chemical groups essential to a variety of RNA and DNA activities.