Blain S W, Goff S P
Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
J Biol Chem. 1993 Nov 5;268(31):23585-92.
RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.
传统上认为核糖核酸酶H（RNases H）仅降解RNA-DNA杂交形式的RNA。我们发现，通过原位凝胶技术检测，野生型莫洛尼鼠白血病病毒（M-MuLV）逆转录酶（RT）能够降解RNA-RNA双链体以及RNA-DNA杂交体中的RNA。在该检测中，大肠杆菌RNase H不会降解RNA-RNA双链体，而双链特异性核糖核酸酶大肠杆菌RNase III则会。M-MuLV RT对RNA-RNA双链体的表观比活性与对RNA-DNA杂交体的相似。单独表达的RT的DNA聚合酶结构域和RNase H结构域均未表现出这种RNA-RNA活性。我们在M-MuLV RT的RNase H结构域中产生了一系列突变，在大肠杆菌中表达突变酶，并检测这些突变体的各种活性。在寡聚（dT）：聚（rA）DNA聚合酶检测中，所有RT的活性都与野生型一样，并且许多都保留了两种核酸酶活性。在RNase H结构域羧基末端有突变的两种酶保留了RNA-DNA活性，但没有RNA-RNA活性。另一种突变酶表现出相反的表型，保留了RNA-RNA核酸酶活性，但没有RNA-DNA核酸酶活性。因此，我们能够从基因上分离这两种活性。这些结果可能有助于确定酶与底物的相互作用。
