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首页> 外文期刊>Molecular and Cellular Biology >Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus
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Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus

机译：酵母mRNA封盖设备的三磷酸酶和鸟苷基转移酶成分之间的遗传，物理和功能相互作用。

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We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical toCET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the γ phosphate of triphosphate-terminated RNA at a rate of 1 s?1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.
机译：我们已经表征了一个基本的酿酒酵母基因 CES5 ，当它以高拷贝存在时，它可以抑制由 ceg1-25 引起的温度敏感型生长缺陷。 em>突变的酵母mRNA鸟嘌呤基转移酶（加帽酶）。 CES5 与 CET1 相同，后者编码酵母加盖装置中的RNA三磷酸酶成分。纯化的重组Cet1以1 s ？1 的速率催化三磷酸封端的RNA的γ磷酸水解。 Cet1是溶液中的单体;它在体外与重组Ceg1结合形成Cet1-Ceg1异二聚体。 Cet1与Ceg1的相互作用引起Ceg1的鸟苷转移酶活性的> 10倍刺激。这种刺激是对GTP底物亲和力增加的结果。截短的蛋白Cet1（201-549）具有RNA三磷酸酶活性，在体外与Ceg1异源二聚体并刺激Ceg1，当以单拷贝形式表达时就足以在体内细胞生长。截短程度更大的衍生物Cet1（246-549）也具有RNA三磷酸酶活性，但不能在体外刺激Ceg1，并且在体内以单拷贝表达时具有致死性。这些数据表明，Cet1-Ceg1相互作用是必不可少的，但不能解决三磷酸酶活性是否也是必需的。哺乳动物的加帽酶Mce1（双功能三磷酸酶-鸟苷基转移酶）在体内替代Cet1。 Mce1的三磷酸酶活性位点半胱氨酸的突变是致命的。因此，RNA三磷酸酶活性对于真核细胞生长至关重要。这项工作强调了通过蛋白质-蛋白质相互作用调节mRNA帽形成的潜力。

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