Tanese N, Goff S P
Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777-81. doi: 10.1073/pnas.85.6.1777.
The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity.
莫洛尼鼠白血病病毒的逆转录酶与所有逆转录病毒的逆转录酶一样，具有能在RNA或DNA模板上进行合成的DNA聚合酶活性，以及对RNA.DNA杂交体形式的RNA具有特异性的核糖核酸酶H活性。我们构建了在细菌中表达的莫洛尼鼠白血病病毒酶的接头插入突变体文库，并对这些突变体的两种酶活性进行了测定。影响DNA聚合酶活性的突变集中在基因5'端近端的三分之二区域，而影响核糖核酸酶H活性的突变则位于其余的3'端三分之一区域。基于这些图谱，制备了分别表达每个结构域的质粒；对这些质粒编码的蛋白质进行的测定表明，每个结构域仅表现出预期的活性。
