Janzen Deanna M, Geballe Adam P
Division of Human Biology, Fred Hutchinson Cancer Research Center, PO Box 19024, 1100 Fairview Avenue North-C2-023, Seattle, WA 98109-1024, USA.
Nucleic Acids Res. 2004 Aug 23;32(15):4491-502. doi: 10.1093/nar/gkh791. Print 2004.
Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.
当终止密码子到达正在翻译的核糖体的A位点时，会发生两种相互竞争的事件，即终止和通读（或无义抑制）。翻译终止导致最终的肽基-tRNA键水解，并释放出完整的新生多肽。或者，通读是指终止密码子被抑制性或近同源转移RNA（tRNA）错误解码，导致翻译越过终止密码子并产生具有C末端延伸的蛋白质。终止与通读的相对频率由终止密码子核苷酸上下文、终止因子的活性和抑制性tRNA的丰度等参数决定。我们结合RNA干扰技术，使用一种灵敏且通用的通读检测方法，评估了真核释放因子1和3（eRF1和eRF3）缺失对人细胞系中终止反应的影响。与eRF1在触发肽基-tRNA水解中已确立的作用一致，我们发现eRF1的缺失增强了293细胞和HeLa细胞中所有三个终止密码子的通读。eRF3在真核生物翻译终止中的作用尚不太清楚，因为其过表达在酵母中已显示具有抗抑制作用，但在哺乳动物系统中并非如此。我们发现，eRF3的缺失对293细胞中的通读几乎没有影响，但确实增加了HeLa细胞中所有三个终止密码子的通读。这些结果支持了eRF3在高等真核生物翻译终止中的直接作用，也突出了终止因子丰度或活性差异以细胞类型特异性方式调节终止与通读反应平衡的可能性。
