在生物體內，Gln-tRNAGln 的合成途徑可分為 direct pathway 與 indirect pathway 兩種。最近的研究發現，在 Saccharomyces cerevisiae 粒線體中， Gln-tRNAGln 的合成是經由 indirect pathway 。首先在 GluRS 的催化下將 Glu 接在 tRNAGln 上，形成了 Glu-tRNAGln 中間產物；然後再經由Glu-tRNAGln amidotransferase (Glu-AdT) 的參與，將 Gln 的醯胺基經由轉胺 作用接到Glu-tRNAGln 的 Glu 上，進而形成 Gln-tRNAGln 。本論文將深入探討 Saccharomyces cerevisiae 的 Glu-AdT，這個酵素是由三個次單元所構成的 GatFAB 複合物。我們想藉由酵母菌雙雜交系統來了解 ScGatFAB 是否跟一般細菌的 GatCAB 結構類似，透過 GatF 做為連接 GatA 與 GatB的橋梁。於是我們接著利用 E. coli overexpression system 想要個別純化出三個蛋白質次單元，以透過 in vitro pull-down assay 進一步確認三者之間的交互作用情形。出乎意料之外的是， 我們發現 ScGatFAB 必須要在 ScGatF、ScGatA 及 ScGatB 三個次單元皆同時存在時，才能形成穩定的結構，這是之前的研究中所沒有提及的。 In general, Gln-tRNAGln formation involves one of pathways, direct or indirect pathway. Recent studies in Saccharomyces cerevisiae have shown that the synthesis of mitochondrial Gln-tRNAGln proceeded through an indirect pathway. First, GluRS mischarges tRNAGln with Glu to form the intermediate, Glu-tRNAGln. Second, glutamyl-tRNAGln amidotransferase (Glu-AdT) transfers the amide group of glutamine to Glu-tRNAGln to form Gln-tRNAGln. We studied the Glu-AdT of Saccharomyces cerevisiae, which is a trimeric GatFAB. By using a yeast two-hybrid system, we wish to analyze the geometry of GatFAB and investigate whether GatF plays an important role in subunit interaction. Next, we want to purify the three subunits of GatFAB by using E. coli overexpression system and identify the interaction more clearly through in vitro pull-down assay. Unexpectedly, the subunits of GatFAB seem to have normal interaction unless three subunits are present at the same time.
