Mutational analysis of the antigenomic trans-acting delta ribozyme:: the alterations of the middle nucleotides located on the P1 stem
Mutational analysis of the antigenomic trans-acting delta ribozyme:: the alterations of the middle nucleotides located on the P1 stem
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DOI：
10.1093/nar/27.6.1473
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发表时间：
1999-03-15
影响因子：
14.9
通讯作者：
Perreault, JP
中科院分区：
文献类型：
--
作者：
Ananvoranich, S;Lafontaine, DA;Perreault, JP
我们以前的报告使用反式作用的反基因组delta核酶和短底物的集合的delta核酶切割表明，中间的核苷酸的pi茎，底物结合位点，是必不可少的切割活性。在这里，我们已经进一步研究了在P1干上的动力学和热力学参数的三角洲核酶切割使用各种核酶变体携带单碱基突变在推定的位置报告的变化的影响。动力学和热力学值的突变研究中获得的两个中间的核苷酸的P1茎表明，结合和活性位点的δ核酶是独特的形成。首先，底物和核酶参与形成螺旋，称为P1茎，其可能包含弱氢键或凸起。第二，三级相互作用，涉及在中间的P1茎的碱基部分可能在定义的化学环境中发挥作用。因此，在途径的后续步骤中，活性位点可能与结合位点同时或随后形成。
Our previous report on delta ribozyme cleavage using a trans-acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the pi stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a consequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.