Basu S, Strobel S A
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520-8114, USA.
Methods. 2001 Mar;23(3):264-75. doi: 10.1006/meth.2000.1137.
Many RNAs, including the ribosome, RNase P, and the group II intron, explicitly require monovalent cations for activity in vitro. Although the necessity of monovalent cations for RNA function has been known for more than a quarter of a century, the characterization of specific monovalent metal sites within large RNAs has been elusive. Here we describe a biochemical approach to identify functionally important monovalent cations in nucleic acids. This method uses thallium (Tl+), a soft Lewis acid heavy metal cation with chemical properties similar to those of the physiological alkaline earth metal potassium (K+). Nucleotide analog interference mapping (NAIM) with the sulfur-substituted nucleotide 6-thioguanosine in combination with selective metal rescue of the interference with Tl+ provides a distinct biochemical signature for monovalent metal ion binding. This approach has identified a K+ binding site within the P4-P6 domain of the Tetrahymena group I intron that is also present within the X-ray crystal structure. The technique also predicted a similar binding site within the Azoarcus group I intron where the structure is not known. The approach is applicable to any RNA molecule that can be transcribed in vitro and whose function can be assayed.
许多RNA，包括核糖体、核糖核酸酶P和II类内含子，在体外活性明确需要单价阳离子。尽管单价阳离子对RNA功能的必要性在超过四分之一个世纪前就已为人所知，但在大型RNA中特定单价金属位点的表征一直难以捉摸。在这里，我们描述了一种生化方法来鉴定核酸中功能重要的单价阳离子。该方法使用铊（Tl+），一种软路易斯酸重金属阳离子，其化学性质与生理碱土金属钾（K+）相似。用硫取代的核苷酸6-硫代鸟苷进行核苷酸类似物干扰图谱分析（NAIM），结合用Tl+对干扰进行选择性金属拯救，为单价金属离子结合提供了独特的生化特征。这种方法在嗜热四膜虫I组内含子的P4-P6结构域内鉴定出一个K+结合位点，该位点也存在于X射线晶体结构中。该技术还预测了结构未知的嗜固氮弧菌I组内含子内的一个类似结合位点。该方法适用于任何可以在体外转录且其功能可以检测的RNA分子。
