The two steps of group II intron self-splicing are mechanistically distinguishable
The two steps of group II intron self-splicing are mechanistically distinguishable
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DOI：
10.1017/s1355838298971643
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发表时间：
1998-08-01
期刊：
影响因子：
4.5
通讯作者：
Padgett, RA
中科院分区：
文献类型：
--
作者：
Podar, M;Perlman, PS;Padgett, RA
由自剪接II组内含子催化的两个酯交换反应发生在两个活性位点或涉及反应基团位置重排的单个活性位点的两种构象中。我们已经研究了由于在5'和3'剪接位点处的非桥氧的硫取代以及在5'剪接位点处的核糖2'羟基的脱氧核糖取代对两个反应的化学步骤速率的影响。这些数据表明，这两个活性位点在与这些基团中的几个基团的相互作用方面有所不同。具体地说，在5'剪接位点的pro-Sp非桥氧的硫取代使第一步分支反应的化学速率降低至少250倍，而在3'剪接位点的pro-Sp氧的取代对第二步分支反应的化学速率仅具有4.5倍的影响。先前的工作表明，在5'和3'剪接位点处的Rp硫代磷酸酯取代将剪接的两个步骤的速率降低到不可检测的水平。这些结果表明，无论是两个不同的活性位点催化的两个步骤，或更显着的改变，必须在一个单一的双功能活性位点，以适应两个不同的反应。
The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.