Cigan A M, Foiani M, Hannig E M, Hinnebusch A G
Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Mol Cell Biol. 1991 Jun;11(6):3217-28. doi: 10.1128/mcb.11.6.3217-3228.1991.
GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.
GCN4是酿酒酵母中氨基酸生物合成基因的转录激活因子，其表达在翻译水平上受氨基酸可用性的调节。GCD1和GCD2是在非饥饿条件下抑制GCN4翻译所必需的负调控因子，这种抑制由GCN4 mRNA前导区的上游开放阅读框（uORF）介导。在氨基酸饥饿的细胞中，GCD因子被正调控因子GCN1、GCN2和GCN3拮抗，从而使GCN4蛋白合成增加。先前的遗传学研究表明，GCD1、GCD2和GCN3在GCN4表达调控中具有密切相关的功能，涉及翻译起始因子2（eIF-2）。与这些预测一致，我们发现GCD1、GCD2和GCN3是一个分子量约为600,000 Da的高分子量复合物的组成成分。这三种蛋白质通过几个生化分级分离步骤共同纯化，并且可以使用针对GCD1或GCD2的抗体进行共免疫沉淀。有趣的是，细胞提取物中存在的一部分eIF-2也与这些调节蛋白共同分级分离和共免疫沉淀，但在0.5 M KCl作用下从GCD1/GCD2/GCN3复合物中解离。将温度敏感型gcdl-101突变体在限制温度下培养，导致多核糖体的平均大小和数量迅速减少，同时无活性的80S核糖体偶联物积累；此外，发现过量的eIF-2α、GCD1、GCD2和GCN3与游离的40S核糖体亚基一起迁移。这些结果表明，GCD1在翻译起始周期的后期步骤中对于涉及eIF-2的基本功能是必需的。我们提出，在氨基酸饥饿的细胞中，降低这种高分子量复合物或eIF-2本身的功能会导致核糖体对uORF的识别减少，以及GCN4起始密码子处的翻译起始增加。我们的结果为如何调节一般起始因子以影响基因特异性翻译控制提供了新的见解。
