Requirements of the secondary structures in the primary transcript for multicopy single-stranded DNA synthesis by reverse transcriptase from bacterial retron-Ec107.
Requirements of the secondary structures in the primary transcript for multicopy single-stranded DNA synthesis by reverse transcriptase from bacterial retron-Ec107.
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发表时间：
1994-05
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通讯作者：
M. Shimada;S. Inouye;M. Inouye
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作者：
M. Shimada;S. Inouye;M. Inouye
多拷贝单链DNA(MsDNA)是由被称为反转录的细菌逆转录元件产生的。它由单链DNA组成，通过2‘，5’-磷酸二酯键与RNA分子的内部G残基相连。已经证明，特定的一级序列以及紧邻G残基下游的二级结构对于cDNA引发反应是必不可少的(Shimamoto，T.，Hsu，M-Y.，Inouye，S.和Inouye，M.(1993)J.Biol)。化学。268、2684-2692)。我们现在已经研究了与DNA相对应的区域中的结构对msDNA合成的要求。由msDNA-Ec107的71个碱基组成的上茎区域被发现不是必需的，该区域可以被删除以有效地产生仅包含36个碱基的单链DNA的截短的msDNA。在下茎区域构建了包括碱基替换、缺失和插入在内的各种突变。研究发现，任何导致更稳定的二级结构的突变都会导致msDNA合成减少。结果表明，逆转录酶需要模板RNA中靠近cDNA启动点的松散的二级结构才能使其延伸。
Multicopy single-stranded DNA (msDNA) is produced by bacterial retroelements called retrons. It consists of single-stranded DNA that is linked to an internal G residue of an RNA molecule by a 2',5'-phosphodiester linkage. It has been demonstrated that specific primary sequences, as well as the secondary structures immediately downstream of the G residue, are essential for the cDNA priming reaction (Shimamoto, T., Hsu, M.-Y., Inouye, S., and Inouye, M. (1993) J. Biol. Chem. 268, 2684-2692). We have now examined the requirement of the structures in the region corresponding to DNA for msDNA synthesis. The upper stem region consisting of 71 bases of msDNA-Ec107 was found not to be essential, and this region could be deleted to efficiently produce a truncated msDNA containing only a 36-base single-stranded DNA. Various mutations including base replacements, deletions, and insertions were constructed in the lower stem region. It was found that any mutations resulting in more stable secondary structures caused reduction in msDNA synthesis. The results indicated that reverse transcriptase requires a loose secondary structure in the template RNA near the cDNA priming site for cDNA elongation.