Shimamoto T, Inouye M, Inouye S
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
J Biol Chem. 1995 Jan 13;270(2):581-8. doi: 10.1074/jbc.270.2.581.
Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.
细菌逆转录酶（RT）负责合成多拷贝单链DNA（msDNA），该单链DNA通过异常的2'，5'-磷酸二酯键与RNA的内部鸟苷残基相连。在这里，我们从携带来自逆转录子-Ec73的RT基因的大肠杆菌中纯化了一种细菌RT，使其达到同质。纯化后的RT-Ec73能够在无细胞系统中使用T7 RNA聚合酶体外产生的RNA模板合成msDNA。体外合成的msDNA只有在用酵母去分支酶DBR1（一种针对2'，5'-磷酸二酯键的特异性核酸酶）处理时才会从模板RNA中释放出来。模板RNA中分支G残基的位置以及无细胞产物的DNA序列与体内合成的msDNA-Ec73的相同。这些结果清楚地表明，msDNA合成中2'，5'-磷酸二酯键的形成是由RT自身完成的。
