Binding stoichiometry of an RNA aptamer and its transcription factor target
Binding stoichiometry of an RNA aptamer and its transcription factor target
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DOI：
10.1006/abio.2002.5710
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发表时间：
2002-07-15
影响因子：
2.9
通讯作者：
Maher, LJ
中科院分区：
文献类型：
--
作者：
Cassiday, LA;Lebruska, LL;Maher, LJ
RNA分子在细胞中起信息、结构和催化作用。RNA还为基因表达修饰剂的设计或遗传选择提供了有趣的原材料。我们一直感兴趣的可能性，天然和/或人工的RNA配体可能被确定为DNA结合蛋白。考虑到这些概念，我们的实验室以前分离出一种31个核苷酸的RNA适体，它特异性地结合到人类转录因子NF-κ B。这种RNA适体（α-p50）在体外竞争性抑制NF-κ B与DNA的结合。适体可以靶向由NF-κ B的两个单体的连接形成的DNA结合沟，可能模拟KB双链体DNA。该模型预测每个NF-κ B二聚体一个RNA适体的结合化学计量。为了验证这一假设，使用了两种互补的生物物理方法。分析性超离心和微电喷雾质谱都表明，每摩尔NF-κ B p50同二聚体结合1摩尔α-p50 RNA。这样的结果与观察到的RNA适体阻断转录因子NF-κ B接近其在DNA上的结合位点的能力一致，并且突出了RNA茎环结构如何模拟DNA双链体的问题。这项工作也证明了质谱法的成功应用，以表征非共价RNA/蛋白质相互作用。(C)2002 Elsevier Science（美国）。
RNA molecules serve informational, structural, and catalytic roles in cells. RNA also offers an interesting raw material for the design or genetic selection of modifiers of gene expression. We have been interested in the possibility that natural and/or artificial RNA ligands might be identified for DNA-binding proteins. With these concepts in mind, our laboratory previously isolated a 31-nucleotide RNA aptamer that specifically binds to human transcription factor NF-kB. This RNA aptamer (alpha-p50) competitively inhibits DNA binding by NF-kB in vitro. The aptamer may target the DNA-binding groove formed by the junction of the two monomers of NF-kB, perhaps mimicking KB duplex DNA. This model predicts a binding stoichiometry of one RNA aptamer per NF-kB dimer. To test this hypothesis, two complementary biophysical methods were utilized. Both analytical ultracentrifugation and microelectrospray mass spectrometry suggest that 1 mol of a-p50 RNA binds per mole of NF-kB p50 homodimer. Such a result is consistent with the observed ability of the RNA aptamer to block the access of transcription factor NF-kB to its binding site on DNA and highlights the question of how an RNA stem-loop structurally mimics a DNA duplex. This work also demonstrates the successful application of mass spectrometry to characterize noncovalent RNA/protein interactions. (C) 2002 Elsevier Science (USA).