HISRS-DOMAIN AND REGULATING GCN2 PROTEIN KINASE

拟议研究的目标是通过以下方式了解机制： 真核细胞调节它们的代谢途径， 生长条件的变化。 在模式系统Saccharomyces 酿酒酵母，饥饿氨基酸诱导GCN 2蛋白激酶 磷酸化真核生物翻译起始的α亚基 因子2（eIF-2）。 该因子的磷酸化刺激 氨基酸转录激活因子GCN 4的翻译 生物合成基因 以前，它表明，序列的 GCN 2的C-末端区域与GCN 2的整个序列同源。 组氨酰-tRNA合成酶（HisRS）。酿酒酵母和人类。 给定 氨酰-tRNA合成酶结合不带电荷的tRNA作为底物， 区分带电荷和不带电荷的tRNA，有人提出， GCN 2中的HisRS相关结构域监测不带电tRNA的浓度 并在细胞内激活相邻的蛋白激酶部分， 饥饿条件下，不带电的tRNA积累。 主 本提案的目的是在净化系统中测试 不带电荷的tRNA水平调节eIF-2-alpha的GCN 2磷酸化。 为了检验这个模型，提出了以下调查路线。 首先，GCN 2在体内监测哪些氨基酸。 已知的是 GCN 4翻译被刺激响应饥饿的任何一个 至少有十种不同的氨基酸 是否每个都需要GCN 2 限制？ 第二，不同物种之间的相互作用 不带电荷的tRNA和GCN 2蛋白激酶将通过体外 结合试验和显示与GCN 2复合的tRNA将被检查 进一步确定它们是否在动力学上增强 eIF-2-alpha通过GCN 2。 最后提出的实验使用遗传工具 可用于分离和表征GCN 2中的突变 改变了底物特异性。 更好地理解GCN 2的调节和底物亲和力 将提供有关哺乳动物应激反应的有用信息， 细胞 已知与GCN 2相关的eIF-2-α激酶抑制 蛋白质合成反应病毒感染（双链RNA 导致DAI激酶的活化）和网织红细胞中的血红素剥夺 （激活HCR蛋白激酶）。

The goal of the proposed research is to understand the mechanisms by which eukaryotic cells regulate their metabolic pathways in response to changes in growth conditions. In the model system Saccharomyces cerevisiae, starvation for amino acids induces the GCN2 protein kinase to phosphorylate the alpha-subunit of eukaryotic translation initiation factor-2 (eIF-2). Phosphorylation of this factor stimulates the translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. Previously, it was shown that the sequence of the C-terminal region of GCN2 is homologous to the entire sequence of histidyl-tRNA synthetase (HisRS) from S. cerevisiae and humans. Given that aminoacyl-tRNA synthetases bind uncharged tRNA as a substrate and distinguish between charged and uncharged tRNA, it was proposed that the HisRS-related domain in GCN2 monitors the concentration of uncharged tRNA in the cell and activates the adjacent protein kinase moiety under starvation conditions when uncharged tRNA accumulates. A primary objective of this proposal is to test in a purified system whether the levels of uncharged tRNA regulate GCN2 phosphorylation of eIF-2-alpha. To test this model the following line of investigation is proposed. First, which amino acids are monitored in vivo by GCN2. It is known that GCN4 translation is stimulated in response to starvation for any one of at least ten different amino acids. Is GCN2 required for each limitation? Secondly, the interaction between different species of uncharged tRNA and GCN2 protein kinase will be measured by in vitro binding assays and the tRNAs shown to complex with GCN2 will be examined further to determine if they kinetically enhance the phosphorylation of eIF-2-alpha by GCN2. The final proposed experiments use genetic tools available in this system to isolate and characterize mutations in GCN2 with altered substrate specificity. A better understanding of the regulation and substrate affinity of GCN2 will provide useful information about stress responses in mammalian cells. It is known that eIF-2-alpha kinases related to GCN2 inhibit protein synthesis in response to viral infection (double-stranded RNA leads to activation of DAI kinase) and heme deprivation in reticulocytes (activates HCR protein kinase).