| 摘要: | 先前研究已知，在Saccharomyces cerevisiae 中，粒腺體以及細胞質功 能的alanyl-tRNA synthetase (稱為AlaRS)是由ALA1 基因所提供。ALA1 基 因利用最靠近mRNA 5’ 端的ATG (1)轉譯出細胞質所需的AlaRS，再利用 ATG1 上游的二個重複ACG (即ACG (-25)和ACG (-24)) 做出粒腺體異構 型，主要仍以ACG (-25)為主。本篇論文著重於酵母菌中使用non-ATG 作為 轉譯起始密碼相關機制的研究。我們以ALA1 為對象去篩選在自然的情況 下，是否有其它的密碼可以取代原本提供粒腺體功能的轉譯起始密碼 (ACG (-25))，篩選結果發現和高等真核生物一樣，酵母菌可以使用和ATG 只差一個核苷酸的密碼 (ATA, ATU, ATC, GTG, TTG, CTG)作為轉譯起始 點，除此之外，我們也篩選到非常罕見且一般不被認為的轉譯起始點密碼 (AGC, CGC, CAT, CAC)可以取代ACG (-25)。我們利用西方點墨法進一步定 量發現，周圍序列-3 ~ -1 的核苷酸對轉譯效率有非常大的影響；另外我們 發現ACG (-25)下游的一個二級結構對轉譯效率也有非常大的影響，其詳細 機制有待進一步研究。 論文第二部份，著重於酵母菌中valyl-tRNA synthetase (ValRS)附加區段 的研究。我們發現這個附加區段具有非專一性tRNA 鍵結的能力，幫助tRNA 鍵結至valine 的酵素上 (Kd 約為2 μM)，而對此附加區段做部分刪除，例 如Δ32-71，便破壞酵素對tRNA 鍵結的能力及酵素胺醯化的活性。除此之 外，先前研究研究顯示附加區段活化轉錄的活性，而我們發現ValRS 可藉 由附加區段將GFP 送入細胞核中，這些結果顯示ValRS 可能參與細胞核基 因的調控。 Recent studies in Saccharomyces cerevisiae have shown that ALA1 (coding for alanyl-tRNA synthetase) initiates the translation of its mitochondrial isoform from two consecutive ACG triplets, with the first ACG being the more robust. In the work described here we focuse on the mechanism of non-ATG initiation in yeast. To explore if any other non-ATG triplets can serve a similar function, the first and second ACG triplets were replaced by a random triplet and ACC, respectively, and the resultant constructs were screened for candidates expressing the mitochondrial activity. We report here that, in addition to ATG, six common non-ATG initiators (ATA, ATU, ATC, GTG, UTG, and CTG) and four rare non-ATG initiators (AGC, CGC, CAT, and CAC) can each functionally substitute for the ACG initiators. Western blot analysis suggests that initiating activity of the non-ATG initiators is significantly affected by the nucleotides at their relative positions -3 ~ -1. Furthermore, a putative pseudoknot structure 17-nucleotide downstream of the non-AUG initiation site also appears to be important for the initiation activity. A second part of the thesis is focused on the valyl-tRNA synthetase (ValRS) appended domain (ad) of Saccharomyces cerevisiae. We show here that this domain is a non-specific tRNA-binding domain (Kd ~ 2 μM) that contributes significantly to the tRNA-binding activity of the valine enzyme. Thus, even a small deletion in the appended domain, such as Δ32-71, has a devastating effect on the enzyme’s tRNA-binding and aminoacylation activities. In addition we show here that the ad can deliver a green fluorescent protein in to the nucleus, further reinforcing the notion that ValRS could be involved regulation of nuclear gene. |
