Ke N, Gao X, Keeney J B, Boeke J D, Voytas D F
Department of Zoology and Genetics, Iowa State University, Ames, 50011, USA.
RNA. 1999 Jul;5(7):929-38. doi: 10.1017/s1355838299990015.
Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.
逆转录转座子和逆转录病毒通过mRNA中间体的逆转录进行复制。大多数逆转录元件从宿主编码的tRNA引物起始逆转录。DNA合成通常从受体茎的3'-OH延伸，该受体茎与逆转录元件mRNA上的序列互补（引物结合位点，PBS）。然而，对于一些逆转录转座子，包括酵母Ty5元件，起始甲硫氨酸tRNA（IMT）的反密码子茎环中的序列与PBS互补。我们利用酵母系统的遗传易处理性来研究Ty5引发的机制。我们发现，破坏与IMT互补性的Ty5 PBS中的突变使转座频率至少降低了800倍。同样，反密码子茎环中的IMT突变使转座至少降低了200倍。Ty5 PBS和IMT之间的碱基配对对于转座至关重要，因为恢复两个突变RNA之间碱基配对的补偿性变化显著恢复了转座。对12个在互补区域之外发生碱基变化的imt突变体的分析未能鉴定出对转座重要的其他tRNA残基。此外，用来自粟酒裂殖酵母和拟南芥的异源IMT进行的试验表明，反密码子茎环之外的残基对转座的影响至多为五倍。我们的遗传系统应该能够进一步确定引发所需的成分，并了解Ty5新型引物产生的机制。
