Kufel J, Kirsebom L A
Department of Microbiology, Biomedical Center, Uppsala, Sweden.
J Mol Biol. 1994 Dec 16;244(5):511-21. doi: 10.1006/jmbi.1994.1749.
We have studied cleavage site selection by M1 RNA, the catalytic subunit of Escherichia coli RNase P, under various reaction conditions using tRNA precursors which are cleaved at two positions. Our results showed that the preference of cleavage site changed with variations in pH or Mg2+ concentration. By contrast, no difference in cleavage site selection was observed with increasing pH in the presence of Ca2+ as the only divalent metal ion. Depending on the identity of the nucleotide at position "+ 72", replacement of Mg2+ with Ca2+ resulted in a change of the main cleavage site irrespective of pH. We conclude that cleavage in the presence of Ca2+ compared to cleavage in the presence of Mg2+ has different structural requirements at and near the cleavage site. UV cross-linking revealed that close points between M1 RNA and its substrate were the same irrespective of pH or the identity of the divalent cation. Our results also showed that the observed pH effects are due to changes in the catalytic cleavage rates rather than to global, structural rearrangements. These data are discussed in terms of metal ion binding near the cleavage sites in the enzyme-substrate complex.
我们使用在两个位置被切割的tRNA前体，研究了大肠杆菌核糖核酸酶P的催化亚基M1 RNA在各种反应条件下的切割位点选择。我们的结果表明，切割位点的偏好会随着pH值或Mg2+浓度的变化而改变。相比之下，在仅存在Ca2+作为唯一二价金属离子的情况下，随着pH值升高，未观察到切割位点选择上的差异。根据“+ 72”位核苷酸的特性，无论pH值如何，用Ca2+取代Mg2+都会导致主要切割位点发生变化。我们得出结论，与在Mg2+存在下的切割相比，在Ca2+存在下的切割在切割位点及其附近具有不同的结构要求。紫外线交联显示，无论pH值或二价阳离子的特性如何，M1 RNA与其底物之间的紧密接触点都是相同的。我们的结果还表明，观察到的pH值效应是由于催化切割速率的变化，而不是由于整体结构重排。这些数据将根据酶 - 底物复合物中切割位点附近的金属离子结合情况进行讨论。
