Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase.
Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase.
复制标题
小插入对 HIV-1 逆转录酶的 RNA 依赖性 DNA 聚合酶活性的影响。
DOI：
10.1016/0042-6822(89)90389-9
复制
发表时间：
1989
期刊：
影响因子：
3.7
通讯作者：
Hughes,SH
中科院分区：
文献类型：
--
作者：
Hizi,A;Barber,A;Hughes,SH
我们已经描述了一种大肠杆菌菌株，它表达高水平的酶活性、可溶性HIV-1逆转录酶（A.希济角麦吉尔和S. H. Hughes，Proc. Natl. Acad. Sci. USA，85，1218-1222，1988）。该克隆可用作酶的来源，并产生和表征逆转录酶中的突变。我们已经在编码逆转录酶的区域中进行了一系列小的框内插入。当突变质粒重新导入E.在大肠杆菌中，它们诱导酶的突变形式的合成。除了一个有趣的例外，在突变体中观察到的RNA依赖性DNA聚合活性的降低与各种逆转录酶之间的序列保守程度密切相关。插入到进化上保守的区域比插入到不太保守的区域对RNA依赖的DNA聚合酶活性有更深远的影响。这种简单相关性的例外是，在编码RNase H的区域中插入一个小片段，产生一种基本上没有RNA依赖性DNA聚合酶活性的蛋白质。我们认为这种突变可能会影响逆转录酶正确折叠的能力，这可能解释了我们以前的观察结果，即小的羧基末端缺失深刻影响RNA依赖的DNA聚合酶活性。
We have described a strain ofEscherichia colithat expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes,Proc. Natl. Acad. Sci. USA, 85, 1218–1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced intoE. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent DNA polymerase activity.