Mei R, Herschlag D
Department of Biochemistry, Beckman Center B400, Stanford University, California 94305-5307, USA.
Biochemistry. 1996 May 7;35(18):5796-809. doi: 10.1021/bi9527653.
Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.
嗜热四膜虫前体核糖体RNA的自我剪接过程包括两个连续的磷酸酯转移步骤。这两个步骤的一个主要区别在于，第一步是外源鸟苷（G）结合到活性位点，而第二步是内含子的3'-末端G414残基结合。第一步在L-21ScaI核酶的研究中已得到广泛表征，该核酶使用外源G作为亲核试剂。在本研究中，通过使用L-21G414核酶研究了第二步所涉及的机制特征。对L-21G414反应的两个方向都进行了研究，在第二步中G414作为离去基团，而在其逆反应中作为亲核试剂。化学步骤的速率常数在外源G结合到L-21ScaI核酶以及L-21G414核酶的分子内鸟苷残基时是相同的。该结果支持了先前提出的单G结合位点模型，并进一步表明在剪接反应的两个步骤中，结合的G的取向和整体活性位点结构是相同的。还提出了在第一步中使用外源G的进化原理。结果表明，L-21G414核酶主要以3'-末端G414对接至G结合位点的形式存在。当G414连接到吸电子的对氨基苯甲酰基时，这种对接会不稳定约100倍。核酶反应的内部平衡K(int)=0.7表明结合的底物和产物在热力学上是匹配的，并且与活性位点内的一定程度的对称性一致。这些观察结果与活性位点中存在第二个镁离子是一致的。最后，相对于连接的外显子类似物，5'外显子类似物从L-21G414核酶的缓慢解离表明了一种确保外显子有效连接的动力学机制，并对整体自我剪接反应提出了新的问题。
