Yang J, Mohr G, Perlman P S, Lambowitz A M
Departments of Molecular Genetics, Biochemistry and Medical Biochemistry, The Ohio State University, 484 West Twelfth Avenue, Columbus, Ohio, 43210, USA.
J Mol Biol. 1998 Sep 25;282(3):505-23. doi: 10.1006/jmbi.1998.2029.
The retrohoming of the yeast mtDNA intron aI1 occurs by a target DNA-primed reverse transcription (TPRT) mechanism in which the intron RNA reverse splices directly into the recipient DNA and is then copied by the intron-encoded reverse transcriptase. Here, we carried out biochemical characterization of the intron-encoded reverse transcriptase and site-specific DNA endonuclease activities required for this process. We show that the aI1 reverse transcriptase has high TPRT activity in the presence of appropriate DNA target sites, but differs from the closely related reverse transcriptase encoded by the yeast aI2 intron in being unable to use artificial substrates efficiently. Characterization of TPRT products shows that the fully reverse spliced intron RNA is an efficient template for cDNA synthesis, while reverse transcription of partially reverse spliced intron RNA is impeded by the branch point. Novel features of the aI1 reaction include a prominent open-circular product in which cDNAs are incorporated at a nick at the antisense-strand cleavage site. The aI1 endonuclease activity, which catalyzes the DNA cleavage and reverse splicing reactions, is associated with ribonucleoprotein particles containing the intron-encoded protein and the excised intron RNA. As shown for the aI2 endonuclease, both the RNA and protein components are used for DNA target site recognition, but the aI1 protein has less stringent nucleotide sequence requirements for the reverse splicing reaction. Finally, perhaps reflecting this relaxed target specificity, in vitro experiments show that aI1 can reverse splice directly into ectopic mtDNA transposition sites, consistent with the previously suggested possibility that this mechanism is used for ectopic transposition of group II introns in vivo.
酵母线粒体DNA内含子aI1的归巢是通过靶DNA引发的逆转录（TPRT）机制实现的，在该机制中，内含子RNA直接反向剪接进入受体DNA，然后由内含子编码的逆转录酶进行复制。在这里，我们对该过程所需的内含子编码的逆转录酶和位点特异性DNA内切酶活性进行了生化特性分析。我们发现，在存在合适的DNA靶位点时，aI1逆转录酶具有较高的TPRT活性，但与酵母aI2内含子编码的密切相关的逆转录酶不同，它不能有效地使用人工底物。对TPRT产物的特性分析表明，完全反向剪接的内含子RNA是cDNA合成的有效模板，而部分反向剪接的内含子RNA的逆转录则受到分支点的阻碍。aI1反应的新特征包括一种突出的开环产物，其中cDNA在反义链切割位点的切口处掺入。催化DNA切割和反向剪接反应的aI1内切酶活性与含有内含子编码蛋白和切除的内含子RNA的核糖核蛋白颗粒相关。正如对aI2内切酶的研究所示，RNA和蛋白质成分都用于DNA靶位点识别，但aI1蛋白对反向剪接反应的核苷酸序列要求较低。最后，也许反映了这种宽松的靶标特异性，体外实验表明，aI1可以直接反向剪接进入异位线粒体DNA转座位点，这与之前提出的该机制在体内用于II类内含子异位转座的可能性一致。
