Abastado J P, Miller P F, Jackson B M, Hinnebusch A G
Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mol Cell Biol. 1991 Jan;11(1):486-96. doi: 10.1128/mcb.11.1.486-496.1991.
GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.
GCN4编码酿酒酵母中氨基酸生物合成基因的转录激活因子，其在翻译水平上受mRNA前导序列中的上游开放阅读框（uORF）调控。uORF4（从5'端开始计数）足以在非饥饿条件下抑制GCN4；uORF1是克服uORF4的抑制作用并在氨基酸饥饿细胞中刺激GCN4翻译所必需的。在uORF4周围插入具有形成二级结构潜力的序列会消除去抑制作用，这表明核糖体通过穿越uORF4序列而非内部结合到GCN4起始位点来到达GCN4。通过证明即使uORF4延长至与GCN4重叠130个核苷酸时仍发生野生型调控，我们提供了有力证据，即那些翻译GCN4的核糖体是通过忽略uORF4的AUG起始密码子来实现的。这一结论与以下事实一致：在去抑制的gcd1突变体中，uORF4 - lacZ融合蛋白的翻译低于不可抑制的gcn2菌株。我们还表明，将uORF1与uORF4之间的距离增加到将uORF1与GCN4分开的野生型间距，会特异性损害uORF1去抑制GCN4翻译的能力。正如预期的那样，这种改变导致gcd1细胞中uORF4 - lacZ翻译增加。我们的结果表明，在饥饿条件下，翻译uORF1的相当一部分核糖体在扫描到uORF4时未能重新组装重新起始所需的因子，但在扫描到GCN4的额外序列后变得有能力重新起始。在非饥饿条件下，核糖体将从uORF1翻译中更快恢复，导致它们全部在uORF4而非GCN4处重新起始。
