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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >RNA from the 5 ' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site
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RNA from the 5 ' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site

机译：R2反转录转座子5'端的RNA控制R2蛋白与其DNA靶位点的结合和裂解

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摘要

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.
机译：非LTR反转录转座子通过靶标引发的逆转录（TPRT）插入真核基因组，在该过程中，裂解的DNA靶标用于引发元件RNA转录物的逆转录。这些元素整合途径中的许多步骤都可以在体外针对R2元素进行表征，因为R2对其DNA靶标及其RNA模板均具有严格的序列特异性。 R2逆转座涉及与插入位点上游和下游的不同DNA序列结合的R2蛋白的相同亚基。发现调节该蛋白质采用哪种DNA结合构象的关键决定因素是从R2元件的5'末端附近的320-nt RNA序列。在缺少该5'RNA的情况下，当存在包含R2转录本3'非翻译区的RNA时，R2蛋白结合插入位点上游的DNA序列，切割第一条DNA链并进行TPRT。在存在320-nt 5'RNA的情况下，R2蛋白与插入位点下游的DNA序列结合。直到从该亚基中除去5'RNA后，似乎不会发生下游亚基对第二条DNA链的切割。我们推测5'RNA的去除通常发生在逆转录过程中，因此提供了在R2逆转录转座反应中第一链和第二链DNA切割的关键时间联系。

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