Kiktev Denis, Moskalenko Svetlana, Murina Olga, Baudin-Baillieu Agnès, Rousset Jean-Pierre, Zhouravleva Galina
Department of Genetics and Breeding, St Petersburg State University, Universitetskaya emb. 7/9, 199034 St Petersburg, Russia.
Mol Genet Genomics. 2009 Jul;282(1):83-96. doi: 10.1007/s00438-009-0447-5. Epub 2009 Apr 16.
The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.
导致必需基因中无义突变不致死的机制目前尚不清楚。在此，我们聚焦于影响在编码翻译终止因子eRF1的必需基因SUP45中携带提前终止密码子（PTC）的酵母细胞生存能力的因素。我们使用双报告系统比较了SUP45基因天然终止密码子的通读效率、自发的sup45 - n（无义）突变、通过定点诱变获得的无义突变（76Q→TAA、242R→TGA、317L→TAG）。结果表明，SUP45基因中的无义突变处于通读效率的中等环境中。我们发现，sup45突变体中存在的一些突变的通读效率与全长Sup45蛋白量无关。这是由于sup45 mRNA稳定性的改变（在sup45 - n突变体中变化3倍）以及突变型Sup45蛋白的降解速率所致。我们的结果表明，PTC位置的一些替换会降低Sup45的稳定性。因此，sup45无义突变体的生存能力受到多种机制的支持，这些机制控制着细胞中功能性Sup45的最终量。
