Section on Nutrient Control of Gene Expression, Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2023 Apr 18;120(16):e2300521120. doi: 10.1073/pnas.2300521120. Epub 2023 Apr 12.
The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator . Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNA, and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl-tRNA ternary complex.
一般氨基酸控制是一种保守的氨基酸饥饿反应，涉及蛋白激酶 Gcn2 的激活，其磷酸化真核起始因子 2（eIF2α），从而抑制全球蛋白质合成并增加酵母转录激活物的翻译。Gcn2 可以被氨基酸饥饿或使延伸核糖体停滞而不减少 tRNA 的氨酰化的条件激活，但尚不清楚这两种情况下是否存在不同的分子机制。我们通过非饥饿依赖性（SI）核糖体停滞鉴定了三种激活酵母细胞中 Gcn2 的状态：用替加环素处理、消除唯一编码 tRNA 的基因和耗尽翻译终止因子 eRF1。我们进一步证明了 Gcn2 的 tRNA 和核糖体结合结构域、正效应蛋白 Gcn1/Gcn20 以及至少两种不同 P1/P2 异二聚体之一与核糖体 P stalk 的 uL10 亚基的连接对于通过 SI-核糖体停滞检测到的 Gcn2 激活是必需的。值得注意的是，对于由组氨酸或支链氨基酸异亮氨酸/缬氨酸饥饿引起的强烈 Gcn2 激活，不需要连接到 P1/P2 蛋白。这些结果表明，Gcn2 激活对 P stalk 的要求因核糖体停滞的方式而异。我们提出，在氨基酸饥饿的细胞中去酰化 tRNA 的积累可以在功能上替代 P stalk 与 Gcn2 的组氨酸-tRNA 合成酶样结构域结合，用于核糖体停滞在缺乏 eEF1A∙GTP∙氨酰-tRNA 三元复合物的 A 位时 eIF2α 激酶的激活。
