Hannig E M, Hinnebusch A G
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Mol Cell Biol. 1988 Nov;8(11):4808-20. doi: 10.1128/mcb.8.11.4808-4820.1988.
GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally. A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.
GCN4编码酿酒酵母中氨基酸生物合成基因的转录激活因子。GCN3产物是氨基酸饥饿细胞中GCN4蛋白合成增加所需的正调控因子。在饥饿条件下，GCN3似乎通过拮抗GCD编码的GCN4表达负调控因子来间接发挥作用；然而，在非饥饿条件下，GCN3也能抑制gcd12突变的影响。这些结果表明，GCN3产物可以根据氨基酸的可用性促进GCN4表达的抑制或激活。我们给出了GCN3基因及其转录本的完整物理描述，以及在不同生长条件下GCN3在转录和翻译水平上的表达测量。GCN3编码一个305个氨基酸的多肽，与任何其他已知蛋白质序列均无明显同源性。GCN3 mRNA不含前导AUG密码子，在GCN3 5'非编码DNA中未发现潜在的GCN4结合位点。与一般控制系统中其他基因的这些调控序列缺失一致，GCN3 mRNA和GCN3 - lacZ融合酶在饥饿和非饥饿条件下的水平相似。这些数据表明，GCN3调控功能对氨基酸可用性的调节发生在翻译后。gcn3缺失在gcd1 - 101突变体中导致无条件致死，支持了GCN3在正常生长条件下表达并在这些情况下与GCD1产物协同执行基本细胞功能的观点。我们描述了一个点突变，该突变在GCN3的羧基末端添加了三个氨基酸，这使其在饥饿条件下所需的正调控功能失活，而不损害其在非饥饿条件下促进GCD12执行功能的能力。
