PDF(Pubmed)
Abstract：
Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.
摘要：
翻译保真度依赖于氨基酰基-tRNA合成酶(AARSs)对转移RNA(tRNA)的准确氨基酰化。AARSs特异于丙氨酸（Ala），亮氨酸(Leu),丝氨酸,和吡咯赖氨酸不识别反密码子碱基。其同源tRNA中的单核苷酸反密码子变体可导致误译。人类基因组包括罕见和更常见的误译tRNA变体。我们研究了三种罕见的人类tRNALeu变体，它们在苯丙氨酸或色氨酸密码子处错误掺入了Leu。在正常条件下或在蛋白酶体抑制的情况下，神经母细胞瘤细胞中每个tRNALeu反密码子变体的表达都会导致荧光蛋白产生的缺陷，而不会显着增加细胞毒性。使用tRNA测序和质谱，我们证实了每个tRNALeu变体都被表达并产生了与Leu的误译。为了探究整个遗传密码对Leu错误合并的灵活性，我们创建了64个酵母菌株,在多西环素诱导系统中表达所有可能的tRNALeu反密码子变体.虽然一些变体显示轻度或无生长缺陷，许多反密码子变体，在35和36位富含G/C，包括用脯氨酸代替Leu，精氨酸丙氨酸,或者甘氨酸,导致经济增长大幅下降。观察到具有同义反密码子的tRNALeu突变体和具有相同反密码子的不同tRNALeu异受体的差异表型缺陷。与tRNAAla反密码子变体的比较表明,在几乎每个密码子处,Ala错误掺入比Leu更可耐受。数据表明，氨基酸取代的性质，tRNA基因，和反密码子都是影响细胞耐受误译tRNA能力的重要因素。
