Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate.
Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate.
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DOI：
10.1111/j.1432-1033.1995.0557d.x
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发表时间：
1995-08
期刊：
影响因子：
--
通讯作者：
E. Kanaya;S. Kanaya
中科院分区：
文献类型：
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作者：
E. Kanaya;S. Kanaya
大肠杆菌核糖核酸酶HI主要识别裂解位点前的DNA/RNA杂交区。为了更详细地了解酶与底物之间的相互作用，使用一系列寡聚DNA/RNA杂交体作为底物研究了酶的动力学性质以及其在基本突起中具有突变的变体。通过将12-B RNA（5 '-CGGAGAUGACGG-3'）与大小和序列不同的DNA寡聚体杂交来制备这些底物。与互补的12-B DNA杂交的12-B RNA主要在A9-C10处被切割。由于使用较长的DNA/RNA底物实现的切割位点和DNA/RNA杂合体的5'末端之间的RNA长度的增加并不严重影响酶的动力学参数，因此12-bp DNA/RNA杂合体似乎足够大以接触酶的整个底物结合位点。这里提出的动力学数据表明，与位于切割位点上游6或7个残基的RNA残基互补的DNA残基与酶的基本突起相互作用，而不管它是否与RNA链杂交。只有当酶或底物或两者的构象在结合时发生变化时，才允许这种相互作用。
Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.