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首页> 外文期刊>Molecular and Cellular Biology >Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.
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Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.

机译：RNA模板上的下游28S基因序列影响引物的选择和R2逆转录酶引发的准确性。

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R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.
机译：R 2非长末端重复逆转座子可插入昆虫28S rRNA基因的独特位点。 R2的单个开放阅读框编码的蛋白质能够进行体外整合的初始步骤。该蛋白质在28S靶DNA的非编码链（用作RNA合成模板的链）上形成切口，并使用通过该切口暴露的3'羟基引发R2 RNA模板的逆转录。此靶标引发的逆转录（TPRT）反应要求RNA模板包含R2元件的250个核苷酸的3'非翻译区。如果此RNA模板在R2元件的精确3'末端终止，则在cDNA合成之前，将额外的核苷酸（我们称为非模板核苷酸）添加至靶标。 RNA模板上下游28S基因序列的存在降低了总效率，但消除了这些非模板化的添加，导致几乎90％的TPRT产物均能重现体内3'连接。在此体外TPRT反应中，最有效地使用28S序列的5至10个核苷酸的模板。对下游28S rRNA序列的需求可能可以解释为什么大多数昆虫的R2元素与大多数非长末端重复序列反转录转座子不同的原因在于，它们在与目标DNA的3'连接处不包含富含A的重复序列。这些体外R2模板上下游序列的存在还表明，R2逆转录酶可以通过使用另一个RNA分子的3'末端引发cDNA合成。这种由RNA引发的cDNA合成不是基于RNA引物和R2模板之间的序列互补性。还已经发现使用非互补RNA分子的3'末端的能力与Neurospora crassa的线粒体Mauriceville质粒的逆转录酶相同。

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