Evans D B, Fan N, Swaney S M, Tarpley W G, Sharma S K
Biochemistry Research, Upjohn Laboratories, Kalamazoo, Michigan 49001.
J Biol Chem. 1994 Aug 26;269(34):21741-7.
An active p15 RNase H domain, consisting of amino acids 427-560 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatography. The purified p15 RNase H domain exhibited no substrate preference for [3H]poly(rG).poly(dC) compared to [3H]poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate. Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant p15 RNase H domain was relatively nonprocessive in RNA degradative activity of the [3H]poly(rA).poly(dT) duplex. Kinetic studies using p15 RNase H showed substrate inhibition with an apparent K(i) value of 0.12 micron for the [3H]poly(rA).poly(dT) hybrid. Substrate inhibition was not observed for the HIV-1 RT-associated RNase H. The results show that the isolated p15 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H.
一个活性p15核糖核酸酶H结构域，由人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的427至560位氨基酸组成，并带有一个基因工程化的五组氨酸N端亲和标签，在大肠杆菌中表达，并通过固定化金属亲和层析纯化至表观均一。与HIV-1 RT相关的核糖核酸酶H相比，纯化后的p15核糖核酸酶H结构域对[3H]聚（rG）·聚（dC）没有底物偏好，而HIV-1 RT相关的核糖核酸酶H对前者底物表现出30倍的偏好。与HIV-1 RT相关的核糖核酸酶H不同，当用未标记的底物进行挑战时，重组p15核糖核酸酶H结构域在[3H]聚（rA）·聚（dT）双链体的RNA降解活性方面相对缺乏连续性。使用p15核糖核酸酶H的动力学研究表明，对于[3H]聚（rA）·聚（dT）杂交体存在底物抑制，表观K（i）值为0.
