| Abstract: | 中文摘要 GRS1是目前酵母菌（Saccharomyces cerevisiae）中唯一已知具功能的Glycyl-tRNA synthetase（GlyRS）基因，雖然在此基因的5`端只有一個ATG轉譯起始點，它卻能同時提供細胞質及粒腺體所需的GlyRS活性。我們的實驗結果顯示，GRS1基因可以同時轉譯出兩個異構型蛋白質：一個轉譯自ATG1，另一個則轉譯自上游相同讀碼框的non-ATG密碼（即TTG -23）；前者作用於細胞質內，後者則作用於粒腺體中。利用5’RACE（rapid amplification of 5’ cDNA ends）與西方點漬法的分析，我們發現這兩個異構型蛋白質是利用leaky scanning的方式，由同一條mRNA上的兩個不同轉譯起始點轉譯而來，這條mRNA的5`端位在相對於ATG1的上游核苷酸-88的位置。雖然這兩種異構型蛋白質有幾乎相同的序列，但因為存在的胞器不同，故在功能上不能互相取代。此外，在融合蛋白的實驗當中，我們發現GRS1的前序列可以解碼出一段胜肽鏈，進而將一個原本屬於細胞質的蛋白質送入粒腺體中，同樣的，GRS1的細胞質異構型蛋白質也可在接上一個外來的粒腺體標的訊號後被送入粒腺體中，這些結果表示GRS1的前序列本身即可解碼出一個完整的粒腺體標的訊號。 在本論文的第二部分，我們著重於酵母菌中使用non-ATG作轉譯起始點的相關機制。利用回報基因分析（reporter gene assay），我們發現兩個並列的non-ATG（ACG-25ACG-24）比一個non-ATG的轉譯效率高，並可取代回報基因的ATG起始點，這是一個從來沒有被發現過的全新機制，表示酵母菌甚或真核細胞可以利用兩個並列的non-ATG來增強原本較差的轉譯起始效率。此外，我們發現在GRS1 TTG-23的下游伴隨一個穩定的RNA二級結構，當我們在此轉譯起始點與二級結構之間加入一段序列時，TTG-23便無法被有效辨識，表示non-ATG存在的位置是被辨識的關鍵因素。另一方面，我們利用隨機突變的方式來篩選中任何可以充當轉譯起始點的密碼，結果發現酵母菌其實跟高等生物一樣可以使用多種跟ATG只差一個核苷酸的non-ATG密碼作為轉譯起始點，但讓人驚訝的是，我們的突變篩選中發現”AGC”竟然也可以當作一個轉譯起始點，據我們所知，這將是第一個在生物中發現的例子：一個跟ATG相差兩個核苷酸的密碼竟可以當作一個轉譯起始點。 ABSTRACT GRS1 was previously identified as the only gene coding for glycyl-tRNA synthetase activity in the yeast Saccharomyces cerevisiae. Evidence presented here shows that two distinct protein isoforms are generated from this gene: a short cytoplasmic form, which is translationally initiated at the putative ATG initiator (i.e., ATG1), and a longer mitochondrial form, which is initiated at an upstream in-frame non-ATG codon (i.e., TTG-23). A reverse transcription approach in conjunction with Western blot analysis suggests that the isoforms are translated via leaky scanning from a single transcript of this gene, which has its 5’-end located at position –88 relative to ATG1. Although the isoforms have essentially the same polypeptide sequence, they cannot substitute for each other because of different localization. A domain fusion study suggests that the leader peptide of the mitochondrial isoform can direct a cytoplasmic passenger into mitochondria, while the cytoplasmic form of glycyl-tRNA synthetase can be converted into a mitochondrial protein by fusion of a heterologous signal peptide, suggesting that it is the nature of the leader peptide that is responsible for the subcellular localization of the isoforms. A second part of the thesis is focused on the mechanism of non-ATG initiation in yeast. Using a reporter gene assay, we show that redundant non-ATG codons have a higher efficiency of translation initiation than a single non-ATG codon at both qualitative and quantitative levels. Even more remarkably, redundant non-ATG codons can substitute for the initiating activity of the ATG initiator of the reporter gene. Thus, the results present a novel mechanism by which the initiating activity of a weak start site can be significantly improved. As in many cases of non-ATG initiation in higher eukaryotes, a stable RNA secondary structure that is predicted to enhance the initiating activity of the non-ATG initiator is found downstream of the GRS1 TTG-23. Introduction of a short sequence between TTG-23 and the secondary structure impairs its initiating activity, suggesting that the distance between the non-ATG initiator and secondary structure is critical for recognition of the weak start site. Our mutagenesis study further shows that, as in higher eukaryotes, many non-ATG codons that differ from ATG by just one nucleotide can be used as translation initiators in yeast. Most surprisingly, an AGC codon, which differs from ATG by two nucleotides, is also functional as an initiator under the conditions used. To our knowledge, this appears to be the first example where a non-ATG triplet that differs from ATG by two nucleotides can still serve as a translation start site. |
