Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNAiMet primer are required for initiation of reverse transcription in vivo
Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNAiMet primer are required for initiation of reverse transcription in vivo
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DOI：
10.1128/mcb.18.2.799
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发表时间：
1998-02-01
影响因子：
5.3
通讯作者：
Wilhelm, FX
中科院分区：
文献类型：
--
作者：
Friant, S;Heyman, T;Wilhelm, FX
酿酒酵母Ty 1逆转录转座子的逆转录由与Ty 1基因组RNA的5'末端附近的引物结合位点（PBS）配对的tRNA（i）（Met）碱基引发。10个核苷酸的PBS与tRNA（i）（Met）的受体茎的最后10个核苷酸互补。对Ty 1 RNA模板和tRNA（i）（Met）引物之间相互作用的结构探测研究表明，除了PBS和tRNA（i）（Met）的3'端之间的相互作用外，Ty 1 RNA的三个短区域（命名为框0、1和2.1）与tRNA（i）（Met）的T和D茎和环相互作用。为了确定这些序列是否对Ty 1逆转录转座子的逆转录途径重要，测试了突变体Ty 1元件和tRNA（i）（Met）支持转座的能力。我们表明，Ty 1盒和tRNA（i）（Met）的T和D茎和环中的互补序列含有对Ty 1逆转录转座至关重要的碱基，tRNA（i）（Met）和盒0、1或2.1之间1或2bp的破坏显著降低转座水平，恢复引物和模板之间碱基配对的补偿突变恢复转座，对Ty 1病毒样颗粒内部产生的逆转录中间体的分析表明，负链强-终止DNA合成受到破坏Ty 1 RNA和引物tRNA（i）（Met）之间互补性突变的影响。
Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(i)(Met) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA, The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(i)(Met). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(i)(Met) primer showed that besides interactions between the PBS and the 3' end of tRNA(i)(Met), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(i)(Met). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(i)(Met) were tested for the ability to support transposition, We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(i)(Met) contain bases that are critical for Ty1 retrotransposition, Disruption of 1 or 2 bp between tRNA(i)(Met) and box 0, 1, or 2.1 dramatically decreases the level of transposition, Compensatory mutations which restore base pairing between the primer,and the template restore transposition, Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(i)(Met).