8月23日，《核酸研究》(Nucleic Acids Research)在线发表了生化与细胞所王恩多研究组的最新研究成果“利用亮氨酸tRNA基因敲除菌株研究酵母亮氨酸tRNA的体内功能”。
氨基酰-tRNA合成酶（aaRS）催化tRNA的氨基酰化反应，为蛋白质合成提供原料。合成正确的氨基酰-tRNA对保证蛋白质合成的质量控制至关重要。aaRS催化反应的专一性不仅涉及到aaRS，也与tRNA有关。目前，通常采用T7 RNA聚合酶转录和核苷酸定点突变的体外方法研究tRNA的功能。体外转录得到的tRNA无修饰碱基，可能影响tRNA的活性。定点突变得到tRNA变种工作量大、耗时。在体外研究tRNA与aaRS的相互作用的研究受到限制，尤其是酵母亮氨酰-tRNA合成酶与tRNALeu相互作用的研究，由于用体外转录方法得到的酵母tRNALeu活性太低，报道不多。
王恩多研究组的博士研究生黄骞利用同源重组的方法，分别敲除了酵母tRNALeu(GAG)和tRNALeu(UAG)的基因，构建了两株酵母tRNALeu体内基因敲除菌株tl(gag)-Δ1和tl(uag)-Δ1-3。发现酵母tRNALeu(GAG)不是酵母生长的必需tRNA；而酵母tRNALeu(UAG)为酵母生长必需，可以识别四种CUN密码子，首次发现酵母用“超摆动”（superwobbling）阅读亮氨酸密码。他们通过化学诱变tRNALeu(UAG)基因，在菌株tl(uag)-Δ1-3中建立了随机突变库，在基因水平上筛选并发现了若干影响tRNALeu(UAG)转录、与LeuRS的氨基酰和编校功能有关的tRNALeu(UAG)的关键碱基。他们还利用tl(uag)-Δ1-3通过基因定点突变得到带有修饰碱基的酵母tRNALeu(UAG)变种，体外研究了用体内方法筛选到的tRNALeu变种的功能，体内和体外数据相符。
该研究不仅丰富了对酵母tRNALeu功能的认识，也为进一步研究酵母tRNA的结构和功能关系提供了有效的研究系统。
该研究得到国家科技部、国家基金委、中国科学院的资助。（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.
