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.

Arnez, J.G. and Moras, D. 1997. Structural and functional considerations of the aminoacylation reaction. Trends Biochem Sci 22: 211-216.
Bailly, M., Blaise, M., Lorber, B., Becker, H.D., and Kern, D. 2007. The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol Cell 28: 228-239.
Bailly, M., Giannouli, S., Blaise, M., Stathopoulos, C., Kern, D., and Becker, H.D. 2006. A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine. Nucleic Acids Res 34: 6083-6094.
Becker, H.D., Reinbolt, J., Kreutzer, R., Giege, R., and Kern, D. 1997. Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes. Biochemistry 36: 8785-8797.
Burbaum, J.J. and Schimmel, P. 1991. Structural relationships and the classification of aminoacyl-tRNA synthetases. J Biol Chem 266: 16965-16968.
Contamine, V. and Picard, M. 2000. Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast. Microbiol Mol Biol Rev 64: 281-315.
Curnow, A.W., Hong, K., Yuan, R., Kim, S., Martins, O., Winkler, W., Henkin, T.M., and Soll, D. 1997. Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc Natl Acad Sci U S A 94: 11819-11826.
Curnow, A.W., Ibba, M., and Soll, D. 1996. tRNA-dependent asparagine formation. Nature 382: 589-590.
Feldmann, H., Aigle, M., Aljinovic, G., Andre, B., Baclet, M.C., Barthe, C., Baur, A., Becam, A.M., Biteau, N., Boles, E., and et al. 1994. Complete DNA sequence of yeast chromosome II. EMBO J 13: 5795-5809.
Felter, S., Diatewa, M., Schneider, C., and Stahl, A.J. 1981. Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases. Biochem Biophys Res Commun 98: 727-734.
Feng, L., Sheppard, K., Tumbula-Hansen, D., and Soll, D. 2005. Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase. J Biol Chem 280(9): 8150-8155
Frechin, M., Senger, B., Braye, M., Kern, D., Martin, R.P., and Becker, H.D. 2009. Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS. Genes Dev 23: 1119-1130.
Gagnon, Y., Lacoste, L., Champagne, N., and Lapointe, J. 1996. Widespread use of the glu-tRNAGln transamidation pathway among bacteria. A member of the alpha purple bacteria lacks glutaminyl-trna synthetase. J Biol Chem 271: 14856-14863.
Hughes, T.R., Marton, M.J., Jones, A.R., Roberts, C.J., Stoughton, R., Armour, C.D., Bennett, H.A., Coffey, E., Dai, H., He, Y.D., Kidd, M.J., King, A.M., Meyer, M.R., Slade, D., Lum, P.Y., Stepaniants, S.B., Shoemaker, D.D., Gachotte, D., Chakraburtty, K., Simon, J., Bard, M., and Friend, S.H. 2000. Functional discovery via a compendium of expression profiles. Cell 102: 109-126.
Mulero, J.J., Rosenthal, J.K., and Fox, T.D. 1994. PET112, a Saccharomyces cerevisiae nuclear gene required to maintain rho+ mitochondrial DNA. Curr Genet 25: 299-304.
Myers, A.M., Pape, L.K., and Tzagoloff, A. 1985. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J 4: 2087-2092.
Nakai, K. and Horton, P. 1999. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24: 34-36.
Nakamura, A., Sheppard, K., Yamane, J., Yao, M., Soll, D., and Tanaka, I. 2010. Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition. Nucleic Acids Res 38: 672-682.
Nakamura, A., Yao, M., Chimnaronk, S., Sakai, N., and Tanaka, I. 2006. Ammonia channel couples glutaminase with transamidase reactions in GatCAB. Science 312: 1954-1958.
Neupert, W. 1997. Protein import into mitochondria. Annu Rev Biochem 66: 863-917.
Pujol, C., Bailly, M., Kern, D., Marechal-Drouard, L., Becker, H., and Duchene, A.M. 2008. Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants. Proc Natl Acad Sci U S A 105: 6481-6485.
Rampias, T., Sheppard, K., and Soll, D. 2010. The archaeal transamidosome for RNA-dependent glutamine biosynthesis. Nucleic Acids Res.10: 1-10
Roy, H., Becker, H.D., Reinbolt, J., and Kern, D. 2003. When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism. Proc Natl Acad Sci U S A 100: 9837-9842.
Rinehart, J., Krett, B., Rubio, M.A., Alfonzo, J.D., and Soll, D. 2005. Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes Dev 19: 583-592.
Sheppard, K. and Soll, D. 2008. On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE. J Mol Biol 377: 831-844.
Sheppard, K., Yuan, J., Hohn, M.J., Jester, B., Devine, K.M., and Soll, D. 2008. From one amino acid to another: tRNA-dependent amino acid biosynthesis. Nucleic Acids Res 36: 1813-1825.
Tumbula, D.L., Becker, H.D., Chang, W.Z., and Soll, D. 2000. Domain-specific recruitment of amide amino acids for protein synthesis. Nature 407: 106-110.
Wilcox, M. and Nirenberg, M. 1968. Transfer RNA as a cofactor coupling amino acid synthesis with that of protein. Proc Natl Acad Sci U S A 61: 229-236.
Wilcox, M. 1969. Gamma-phosphoryl ester of glu-tRNA-GLN as an intermediate in Bacillus subtilis glutaminyl-tRNA synthesis. Cold Spring Harb Symp Quant Biol 34: 521-528.
Wu, J., Bu, W., Sheppard, K., Kitabatake, M., Kwon, S.T., Soll, D., and Smith, J.L. 2009. Insights into tRNA-dependent amidotransferase evolution and catalysis from the structure of the Aquifex aeolicus enzyme. J Mol Biol 391: 703-716.
Yuan, J., Sheppard, K., and Soll, D. 2008. Amino acid modifications on tRNA. Acta Biochim Biophys Sin (Shanghai) 40: 539-553.
王如玉 (2004) 鑑定酵母菌中具高親和力的tRNA 結合蛋白。中央大學碩士論文
葉曜榮 (2008) 探討酵母菌 Glutaminyl-tRNA synthetase 對於粒腺體功能之影響。中央大學碩士論文