Mitra S, Bechhofer D H
Department of Biochemistry, Mount Sinai School of Medicine of City University New York, New York 10029-6574.
J Biol Chem. 1994 Dec 16;269(50):31450-6.
Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase III-like enzyme called "Bs-RNase III." Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase III, which was purified from a protease-deficient strain of B. subtilis. Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed. The effects of these changes on the rate of Bs-RNase III cleavage were measured. The activity of Bs-RNase III and Escherichia coli RNase III on heterologous substrates was also tested. Although the local environment of the site of Bs-RNase III cleavage appears very similar to that of E. coli RNase III, there are important differences in their substrate specificity.
枯草芽孢杆菌噬菌体SP82编码几种早期RNA，先前已证明这些RNA会被一种名为“Bs-RNase III”的类RNase III酶切割。将编码这些RNA序列的DNA片段克隆到T7 RNA聚合酶启动子下游，从而合成用于测试Bs-RNase III切割特异性的底物，该酶是从枯草芽孢杆菌的蛋白酶缺陷菌株中纯化得到的。构建了切割位点处或其附近的单核苷酸变化以及切割位点上下游的缺失。测量了这些变化对Bs-RNase III切割速率的影响。还测试了Bs-RNase III和大肠杆菌RNase III对异源底物的活性。尽管Bs-RNase III切割位点的局部环境看起来与大肠杆菌RNase III非常相似，但它们的底物特异性存在重要差异。
