GCD10, A TRANSLATIONAL REPRESSOR OF GCN4, IS THE RNA-BINDING SUBUNIT OF EUKARYOTIC TRANSLATION INITIATION FACTOR-III
GCD10, A TRANSLATIONAL REPRESSOR OF GCN4, IS THE RNA-BINDING SUBUNIT OF EUKARYOTIC TRANSLATION INITIATION FACTOR-III
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DOI：
10.1101/gad.9.14.1781
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发表时间：
1995-07-15
影响因子：
10.5
通讯作者：
TAMAME, M
中科院分区：
文献类型：
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作者：
GARCIABARRIO, MT;NARANDA, T;TAMAME, M
GCN 4 mRNA的翻译是通过一种再起始机制进行的，该机制涉及其前导序列中的四个短的上游开放阅读框（uORF）。通过磷酸化降低真核起始因子-2（eIF-2）的活性抑制酵母中的一般翻译，但通过允许核糖体扫描通过uORF并在GCN 4处重新起始而刺激GCN 4表达。GCD 10最初被鉴定为GCN 4的翻译抑制因子。我们在这里表明，GCD 10是一个必需的蛋白质的54.6 kD，是需要在体内启动总蛋白质合成。GCD 10在体外结合RNA，我们提出了强有力的生化证据，它是相同的酵母起始因子-3（eIF-3）的RNA结合亚基。eIF-3是一种多亚基复合物，在体外刺激翻译起始的几个不同步骤。我们认为gcd 10突变降低了eIE-3刺激eIF-2/GTP/Met-tRNA（i）（Met）三元复合物与核糖体小亚基结合的能力。这就解释了为什么eIE-3突变模拟eIF-2 α磷酸化，允许核糖体绕过uORF并在GCN 4重新启动。我们的研究结果表明，GCN 4的表达提供了一个敏感的在体内测定eIE-3的功能，在起始复合物的形成。
GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIE-3 to stimulate binding of eIF-2/GTP/Met-tRNA(i)(Met) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIE-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIE-3 in initiation complex formation.