Austin S A, Pollard J W, Jagus R, Clemens M J
Eur J Biochem. 1986 May 15;157(1):39-47. doi: 10.1111/j.1432-1033.1986.tb09635.x.
The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.
在许多特性明确的含有不同温度敏感型氨酰 - tRNA合成酶的中国仓鼠卵巢（CHO）细胞突变体的提取物中，对多肽链起始的调控进行了研究。当培养物从允许温度34℃转移到非允许温度39.5℃时，这些细胞的起始速率大幅下降。在用[35S]Met - tRNAfMet或[35S]甲硫氨酸短暂孵育期间，暴露于39.5℃的突变细胞系提取物中，天然40S核糖体亚基和80S核糖体上起始复合物的形成受到严重损害。暴露于39.5℃的野生型细胞未显示出对蛋白质合成或起始复合物形成的任何抑制作用。在非允许温度下孵育的突变细胞提取物中，40S起始复合物形成的抑制作用显示与mRNA结合的可能变化或多肽链延伸速率无关，也不是由于起始因子eIF - 2总量的任何减少。然而，对温度敏感突变体核糖体后上清液中eIF - 2·GTP·Met - tRNAfMet三元复合物形成的测定表明，细胞暴露于39.5℃后，eIF - 2的活性存在明显缺陷，并且向在34℃和39.5℃孵育的细胞的无细胞蛋白质合成系统中添加外源eIF - 2消除了它们之间的活性差异。在突变的CHO细胞中，起始因子本身的活性并非直接对温度敏感。结果表明，氨酰 - tRNA合成酶的活性可以影响eIF - 2在完整细胞中结合Met - tRNAfMet并形成40S起始复合物的能力，这表明多肽链延伸和链起始之间存在调控联系。
