Ananvoranich S, Lafontaine D A, Perreault J P
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Nucleic Acids Res. 1999 Mar 15;27(6):1473-9. doi: 10.1093/nar/27.6.1473.
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 P1 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 con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.
我们之前关于使用反式作用基因组δ核酶和一系列短底物进行δ核酶切割的报告表明，P1茎的中间核苷酸（即底物结合位点）对切割活性至关重要。在此，我们进一步研究了P1茎改变对δ核酶切割动力学和热力学参数的影响，使用了在报道的假定位置携带单碱基突变的各种核酶变体。在对P1茎的两个中间核苷酸进行突变研究中获得的动力学和热力学值表明，δ核酶的结合位点和活性位点是独特形成的。首先，底物和核酶参与形成一种螺旋结构，称为P1茎，其可能包含一个弱氢键或一个凸起。其次，涉及P1茎中间碱基部分的三级相互作用可能在确定化学环境中起作用。因此，活性位点可能在该途径的后期步骤中与结合位点同时或随后形成。
