Yang W, Hendrickson W A, Kalman E T, Crouch R J
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
J Biol Chem. 1990 Aug 15;265(23):13553-9.
Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).
来自大肠杆菌的核糖核酸酶H（RNase H）是一种内切核酸酶，可特异性降解RNA:DNA杂交体中的RNA。该酶是一条由155个氨基酸残基组成的单多肽链，其中4个是甲硫氨酸。为了通过多波长反常衍射技术解析大肠杆菌RNase H的晶体三维结构，我们构建了过量表达硒代甲硫氨酰RNase H的大肠杆菌甲硫氨酸营养缺陷型菌株。MIC88每升培养物可产生约10毫克硒代甲硫氨酰RNase H，这与天然重组蛋白的过量表达相当。我们已将这两种蛋白纯化至均一，并在硫酸盐存在下将它们同晶型结晶。这些是空间群为P2(1)2(1)2(1)的I型晶体，晶胞参数为a = 41.8 Å，b = 86.4 Å，c = 36.4 Å，每个不对称单元中有一个单体，溶剂含量约为36%（v/v）。两种蛋白的晶体衍射分辨率超过2 Å布拉格间距，并且在X射线束中相对稳定。用NaCl替代硫酸盐后，天然RNase H的晶体在pH 7.0至8.0之间以I'型（与I型非常相似）生长；在pH 8.8时，在空间群P2(1)2(1)2(1)中获得II型晶体，其a = 44.3 Å，b = 87.3 Å，c = 35.7 Å。II型晶体可通过浸泡在磷酸盐缓冲液中转化为I型。Kanaya等人（Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546 - 11549）也报道了II型的RNase H晶体。
