Strobel S A, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1996 Jan 30;35(4):1201-11. doi: 10.1021/bi952244f.
A phylogenetically conserved guanine.uracil (G.U) pair defines the 5'-exon/intron boundary of precursor RNAs containing group I introns. In this wobble base pair, the G forms two hydrogen bonds with U in a base pairing geometry shifted from that of a canonical Watson-Crick pair. On the basis of thermodynamic measurements of synthetic base pair analogs (inosine, diaminopurine riboside, guanosine, or adenosine paired with U, C, or isocytidine) in place of the G.U pair, we have previously reported that the N2 exocyclic amine of the G is important for docking the 5'-exon into the active site of the Tetrahymena ribozyme [Strobel, S. A., & Cech, T. R. (1995) Science 267, 675-679]. Here we describe kinetic characterization of ribozyme-substrate combinations containing the same series of analogs. By measuring the rate constants of 5'-exon miscleavage (cleavage at incorrect phosphates), we demonstrate that the 5'-exon/intron boundary is primarily defined by the exocyclic amine of the G. The amine makes its contribution (2.5 kcal.mol-1) in the context of all three wobble pairs tested but fails to make a significant contribution (< 0.8 kcal.mol-1) when presented in a Watson-Crick base pairing geometry. We also demonstrate that the exocyclic amine makes a modest contribution to chemical transition state stabilization (1.0 kcal.mol-1 relative to an inosine-U pair). The majority of this transition state contribution (0.7 kcal.mol-1) is independent of that contributed by the 2'-hydroxyl of the neighboring U. This argues against the model in which substantial transition state stabilization is derived from a water molecule bridging between the exocyclic amine of G and the 2'-hydroxyl of U. Instead it suggests that the tertiary interaction between the exocyclic amine and its hydrogen bonding partner in the active site is slightly improved during the chemical transition. We conclude that the exocyclic amine of G is the primary contributor to many characteristics of reactivity that have been ascribed to the conserved G.U pair, including stabilization of the chemical transition state and definition of the 5'-exon/intron boundary.
一种系统发育保守的鸟嘌呤-尿嘧啶（G·U）碱基对定义了含有I组内含子的前体RNA的5'-外显子/内含子边界。在这种摆动碱基对中，G与U形成两个氢键，其碱基配对几何结构与典型的沃森-克里克碱基对不同。基于对合成碱基对类似物（次黄嘌呤、二氨基嘌呤核糖苷、鸟苷或腺苷与U、C或异胞嘧啶配对）取代G·U碱基对的热力学测量，我们之前报道过G的N2外环胺对于将5'-外显子对接至嗜热四膜虫核酶的活性位点很重要[斯特罗贝尔，S. A.，& 切赫，T. R.（1995年）《科学》267，675 - 679]。在此我们描述了含有相同系列类似物的核酶-底物组合的动力学特征。通过测量5'-外显子错切（在不正确的磷酸处切割）的速率常数，我们证明5'-外显子/内含子边界主要由G的外环胺定义。在所有测试的三种摆动碱基对的情况下，该胺都发挥了作用（2.5千卡·摩尔⁻¹），但当以沃森-克里克碱基配对几何结构呈现时，它未能做出显著贡献（< 0.8千卡·摩尔⁻¹）。我们还证明外环胺对化学过渡态稳定有适度贡献（相对于次黄嘌呤-尿嘧啶碱基对为1.0千卡·摩尔⁻¹）。这种过渡态贡献的大部分（0.7千卡·摩尔⁻¹）与相邻U的2'-羟基所做的贡献无关。这与一种模型相悖，该模型认为大量的过渡态稳定是由一个水分子在G的外环胺和U的2'-羟基之间桥接产生的。相反，这表明在化学过渡过程中，外环胺与其在活性位点的氢键伙伴之间的三级相互作用略有改善。我们得出结论，G的外环胺是许多归因于保守G·U碱基对的反应性特征的主要贡献者，包括化学过渡态的稳定以及5'-外显子/内含子边界的定义。
