8月23日，《核酸研究》(Nucleic Acids Research)在線發(fā)表了生化與細(xì)胞所王恩多研究組的最新研究成果“利用亮氨酸t(yī)RNA基因敲除菌株研究酵母亮氨酸t(yī)RNA的體內(nèi)功能”,。
氨基酰-tRNA合成酶（aaRS）催化tRNA的氨基?；磻?yīng)，為蛋白質(zhì)合成提供原料,。合成正確的氨基酰-tRNA對保證蛋白質(zhì)合成的質(zhì)量控制至關(guān)重要,。aaRS催化反應(yīng)的專一性不僅涉及到aaRS，也與tRNA有關(guān),。目前,，通常采用T7 RNA聚合酶轉(zhuǎn)錄和核苷酸定點(diǎn)突變的體外方法研究tRNA的功能。體外轉(zhuǎn)錄得到的tRNA無修飾堿基,，可能影響tRNA的活性,。定點(diǎn)突變得到tRNA變種工作量大、耗時(shí),。在體外研究tRNA與aaRS的相互作用的研究受到限制,，尤其是酵母亮氨酰-tRNA合成酶與tRNALeu相互作用的研究，由于用體外轉(zhuǎn)錄方法得到的酵母tRNALeu活性太低,，報(bào)道不多,。
王恩多研究組的博士研究生黃騫利用同源重組的方法，分別敲除了酵母tRNALeu(GAG)和tRNALeu(UAG)的基因,，構(gòu)建了兩株酵母tRNALeu體內(nèi)基因敲除菌株tl(gag)-Δ1和tl(uag)-Δ1-3,。發(fā)現(xiàn)酵母tRNALeu(GAG)不是酵母生長的必需tRNA；而酵母tRNALeu(UAG)為酵母生長必需,，可以識(shí)別四種CUN密碼子,，首次發(fā)現(xiàn)酵母用“超擺動(dòng)”（superwobbling）閱讀亮氨酸密碼。他們通過化學(xué)誘變tRNALeu(UAG)基因,，在菌株tl(uag)-Δ1-3中建立了隨機(jī)突變庫,，在基因水平上篩選并發(fā)現(xiàn)了若干影響tRNALeu(UAG)轉(zhuǎn)錄、與LeuRS的氨基酰和編校功能有關(guān)的tRNALeu(UAG)的關(guān)鍵堿基,。他們還利用tl(uag)-Δ1-3通過基因定點(diǎn)突變得到帶有修飾堿基的酵母tRNALeu(UAG)變種，體外研究了用體內(nèi)方法篩選到的tRNALeu變種的功能，體內(nèi)和體外數(shù)據(jù)相符,。
該研究不僅豐富了對酵母tRNALeu功能的認(rèn)識(shí),，也為進(jìn)一步研究酵母tRNA的結(jié)構(gòu)和功能關(guān)系提供了有效的研究系統(tǒng)。
該研究得到國家科技部,、國家基金委,、中國科學(xué)院的資助。（生物谷Bioon.com）
doi: 10.1093/nar/gks783
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In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain
Qian Huang, Peng Yao, Gilbert Eriani and En-Duo Wang
The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNALeu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNALeu knockout strains carrying deletions of the genes for tRNALeu(GAG) and tRNALeu(UAG). Disrupting the single gene encoding tRNALeu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNALeu(UAG) had a lethal effect on the yeast strain, indicating that tRNALeu(UAG) decoding capacity could not be compensated by another tRNALeu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNALeu(UAG), a selection to identify critical tRNALeu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.
