Post K, Guo J, Kalman E, Uchida T, Crouch R J, Levin J G
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1993 Jun 1;32(21):5508-17. doi: 10.1021/bi00072a004.
The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes. In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed. These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992) Science 256, 1783-1790). With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer. Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure. The chimeric RT behaved like E. coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA. Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed. The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation. Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.
通过研究AKR鼠白血病病毒（MuLV）酶的活性，对逆转录酶（RT）的聚合酶结构域与核糖核酸酶H（RNase H）结构域之间的功能关系进行了研究。除野生型外，还表达了一种缺失整个RNase H结构域的无RNase H的RT以及另外两种聚合酶:RNase H比例异常的突变体。这些突变体包括：（i）一种嵌合蛋白，其中MuLV的RNase H结构域被整个大肠杆菌RNase H序列取代；（ii）一种RT，在与人类免疫缺陷病毒1型RT的p66亚基中的“连接”亚结构域类似的区域有126个氨基酸缺失（科尔施泰特，L.A.，王，J.，弗里德曼，J.M.，赖斯，P.A.，& 施泰茨，T.A.（1992年）《科学》256卷，1783 - 1790页）。对于野生型RT，主要的RNase H切割反应与DNA合成相协调，发生在与DNA引物3'末端相距15个核苷酸的位置。根据一个关于RNA - DNA杂交底物结合与酶结构的模型，解释了更靠近RNA 5'末端的额外切割。嵌合RT的行为类似于大肠杆菌RNase H，其RNase H活性比野生型RT高300倍，并且其DNA合成能力受到限制。还观察到缺失突变体的聚合酶和RNase H活性的定性和定量变化。RNase H结构域似乎独立于聚合酶结构域发挥作用，这支持了这样一种观点，即两个活性中心之间正常的空间关系被突变破坏了。综上所述，我们的结果表明，如对于具有两个相互依赖结构域的酶所预期的那样，正常聚合酶:RNase H比例的改变可对聚合酶和RNase H切割活性产生深远影响。
