雖然蛋白質轉譯的基本步驟在原核及真核細胞內十分相似，但是轉譯的起始機制卻有明顯的不同：在原核細胞內，30S核醣體會藉由辨認mRNA上的Shine-Dalgarno序列及AUG將核醣體置放於正確的轉譯起始區域；但是在真核細胞內，mRNA上並沒有類似的Shine-Dalgarno序列， 40S核醣體會先結合到mRNA 5’端的一個”cap”結構，然後朝3’端逐步滑行，直到遇見第一個AUG密碼，才開始進行轉譯工作。因此絕大部份的真核細胞mRNA都是以最靠近5’端的第一個AUG作為轉譯起始密碼；只有極少數（< 1%）的哺乳類細胞或病毒mRNA可以使用non-AUG（泛指與AUG只有一個核苷酸差異的密碼）作為轉譯起始密碼，這些non-AUG起始密碼可以藉由周邊序列的幫忙加強其與40S核醣體的交互作用。相對地在低等真核細胞（如酵母菌）內周邊序列似乎沒有類似的增強效果，因此一般認為酵母菌無法使用自然序列上的non-AUG作為轉譯起始點。然而我們最近的實驗證實：在酵母菌內的ALA1（解碼alanyl-tRNA synthetase）及GRS1（解碼glycyl-tRNA synthetase）二個基因不但可以使用傳統的AUG密碼作為其細胞質蛋白質異構型的轉譯起始點，也可以使用其上游相同讀碼框內的一個non-AUG密碼作為其粒線體蛋白質異構型的轉譯起始點，而且不同於以往的認知:周邊序列對non-AUG起始密碼的轉譯效率有非常巨大的影響。這些突破性的新發現在研究酵母菌基因表現上是一個重要的里程碑，然而其詳細的作用機制尚不清楚。本計劃為期三年，在此期限內我們將以ALA1及GRS1為研究主題探討轉譯起始相關機制，重點包括：（1）不同環境或生長條件下，這些雙功能的基因如何調控其細胞質及粒線體蛋白質異構型的相對表現量；（2）以隨機突變及功能性測試篩選出所有可能的non-AUG轉譯起始密碼，並定量分析其使用效率；（3）鑑定及分析參與non-AUG轉譯起始機制的特定RNA二級結構。Although the principles of protein translation are similar in prokaryotes and eukaryotes, the mechanisms that govern the initiation step of protein synthesis differ significantly. In prokaryotes, selection of the initiation codon is dictated by the interactions between the 30S ribosomal subunit and Shine-Dalgarno sequence, while in eukaryotes, translation occurs through a “scanning model” in which the 40S ribosomal subunit first binds to a “cap” structure on the 5’ end of an mRNA, and then scans toward the 3’ end of the molecule until it encounters a suitable AUG codon. Thus, nearly all eukaryotic mRNAs use the AUG closest to the 5’ end as the translational initiator. However, some cases of mammalian cellular and viral mRNAs are capable of using non-AUG codons (that differ from AUG by one nucleotide) as the translation start site. It must be noted, however, that sequence context does not have similar effects in lower eukaryotes (such as yeast); therefore yeast was not previously thought to be capable of non-AUG translation initiation. Contrary to this belief, our recent results suggest that both ALA1 (the yeast gene coding for alanyl-tRNA synthetase) and GRS1 (the yeast gene coding for glycyl-tRNA synthetase) use a naturally occurring non-AUG codon as the alternative translational initiator. In both cases, the cytoplasmic form is translationally initiated from an AUG codon, while its mitochondrial isoform is initiated from an upstream non-AUG codon. Most surprisingly, sequence context drastically affects the initiating activity of the non-AUG initiation codons in yeast. This discovery is a landmark in the study of gene expression and evolution in eukaryotic cells, but the mechanisms that make non-AUG initiation possible in lower eukaryotes remain elusive. The present proposal is a 3-year project in which we propose to use ALA1 and GRS1 as models to elucidate the mechanisms of non-AUG initiation; the main points include: (1) elucidating the mechanism that regulates the differential expression of the cytoplasmic and mitochondrial isoforms of alanyl-tRNA synthetase in response to various stimuli or growth conditions; (2) screening for all possible translational initiation codons in yeast by random mutagenesis in combination with functional assays, and quantification of their relative efficiencies; (3) identification of specific RNA secondary structures important for non-AUG translation initiation. 研究期間 : 9808 ~ 9907
