Miller P F, Hinnebusch A G
Unit on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Genes Dev. 1989 Aug;3(8):1217-25. doi: 10.1101/gad.3.8.1217.
Translational control of the GCN4 gene of Saccharomyces cerevisiae requires at least two of the four short upstream open reading frames (URFs) in the leader of GCN4 mRNA. URF4 is a strong negative element that is sufficient for repression of GCN4 expression in normal growth conditions. URF1 is approximately 30-fold less effective as a translational barrier when it is the single URF present in the mRNA leader and is required upstream from URF4 for efficient derepression of GCN4 expression under amino acid starvation conditions. We show that the last codon plus 10 bp immediately after the stop codon of URF4 are sufficient to convert URF1 into a strong translational barrier when it is present as a solitary URF. This result suggests that the characteristics of translation termination at URF4 are responsible largely for its strong inhibitory effect on translation initiation at the GCN4 AUG codon. Introduction of the same URF4 sequences at URF1 also reduces GCN4 expression under derepressing conditions when URF1 is upstream from URF4. This fact suggests that URF1 translation normally is compatible with efficient scanning and initiation downstream and that this property is required for its ability to overcome the translational barrier at URF4. These findings are consistent with the idea that ribosomes must first translate URF1 and then resume scanning in order to traverse URF4 sequences under starvation conditions. Our results indicate that nucleotides located 3' to the stop codon can be as important as those located 5' to the start site in determining the inhibitory effect of an URF on translation initiation downstream.
酿酒酵母GCN4基因的翻译调控需要GCN4 mRNA前导序列中的四个短上游开放阅读框（URF）中的至少两个。URF4是一个强大的负调控元件，足以在正常生长条件下抑制GCN4的表达。当URF1是mRNA前导序列中唯一存在的URF时，它作为翻译障碍的效率约低30倍，并且在氨基酸饥饿条件下，为了有效解除对GCN4表达的抑制，它在URF4的上游是必需的。我们表明，当URF1单独存在时，URF4终止密码子后的最后一个密码子加上紧接着的10个碱基对足以将URF1转变为一个强大的翻译障碍。这一结果表明，URF4处翻译终止的特征在很大程度上导致了其对GCN4 AUG密码子处翻译起始的强烈抑制作用。当URF1在URF4的上游时，在解除抑制的条件下，在URF1处引入相同的URF4序列也会降低GCN4的表达。这一事实表明，URF1的翻译通常与下游的有效扫描和起始兼容，并且这一特性是其克服URF4处翻译障碍能力所必需的。这些发现与核糖体必须首先翻译URF1然后在饥饿条件下重新开始扫描以便穿过URF4序列的观点一致。我们的结果表明，位于终止密码子3'端的核苷酸在决定URF对下游翻译起始的抑制作用方面可能与位于起始位点5'端的核苷酸同样重要。
