Thompson S R, Gulyas K D, Sarnow P
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):12972-7. doi: 10.1073/pnas.241286698. Epub 2001 Oct 30.
Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.
翻译起始的内部机制可由特定的内部核糖体进入位点（IRES）元件介导，这些元件存在于某些哺乳动物和病毒的mRNA分子中。到目前为止，这些哺乳动物细胞和病毒的IRES元件在酿酒酵母中尚未显示出功能。我们在此报告，最近在蟋蟀麻痹病毒基因组中发现的一个IRES能够指导酿酒酵母中双顺反子mRNA里第二个URA3顺反子的高效翻译，从而赋予细胞不依赖尿嘧啶生长的能力。奇怪的是，该IRES在野生型酵母中功能不佳，但在eIF2激酶GCN2组成型表达的情况下或在两个起始tRNA(met)基因被破坏的细胞中能高效发挥作用。这两种情况都已被证明会降低三元eIF2 - GTP/起始tRNA(met)复合物的数量。此外，在存在依地恩（一种已被证明会干扰携带三元复合物的核糖体亚基对起始密码子识别的化合物）的情况下，从酿酒酵母制备的具有翻译活性的提取物中也观察到了不依赖tRNA(met)的起始现象。因此，蟋蟀麻痹病毒IRES可能在酿酒酵母中通过在缺乏eIF2和起始tRNA(met)的情况下进行内部起始来招募核糖体，其机制与哺乳动物细胞中使用的不依赖因子的核糖体招募机制相同。这些发现将有助于利用酵母遗传学来确定核糖体内部进入的机制。
