Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery
Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery
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DOI：
10.1261/rna.2475211
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发表时间：
2011-03-01
期刊：
影响因子：
4.5
通讯作者：
Lorsch, Jon R.
中科院分区：
文献类型：
--
作者：
Takacs, Julie E.;Neary, Timothy B.;Lorsch, Jon R.
真核生物中的翻译起始涉及十多个蛋白质因子。已发现六个因素的改变会降低酵母中核糖体前起始复合物识别起始密码子的保真度，这种表型称为 Sui(-)。目前尚无已知小分子会影响起始密码子识别的保真度。此类化合物将成为探索翻译起始及其调节的分子机制的有用工具。为了找到具有这种作用的化合物，我们在酿酒酵母中使用双荧光素酶测定建立了高通量筛选。对超过 55,000 种化合物的筛选揭示了两种结构相关的分子，它们在双荧光素酶测定中将起始密码子选择的保真度降低了大约两倍。使用监测非 AUG 起始密码子翻译的其他体内测定证实了这种效应。这两种化合物都增加了天然上游开放阅读框的翻译，先前显示该开放阅读框可在 UUG 处启动翻译。还发现这些化合物会加剧野生型真核起始因子 (eIF) 1 的单倍体不足或 eIF1 突变导致的 UUG 作为起始密码子（Sui(-) 表型）的使用增加。此外，过表达 eIF1 可以抑制化合物的作用，众所周知，eIF1 可以恢复在各种其他起始因子中含有 Sui(-) 突变的菌株中起始密码子选择的保真度。总之，这些数据强烈表明这些化合物影响翻译机制本身，从而降低选择 AUG 作为起始密码子的准确性。
Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.