跳到主要內容

簡易檢索 / 詳目顯示

回結果列表
研究生： 陳順佳
Shun-Jia Chen
論文名稱： 探討酵母菌GlyRS基因的表現及功能
Study of the expression and function of yeast GlyRS genes
指導教授： 王健家
Chien-Chia Wang
口試委員:
學位類別： 博士
Doctor
系所名稱： 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度： 99
語文別： 英文
論文頁數： 122
外文關鍵詞： aminoacyl-tRNA synthetase, inducible gene, sequence context, aminoacylation
相關次數： 點閱：12  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 之前的研究發現釀酒酵母中ALA1及GRS1基因都是使用non-AUG起始密碼轉譯出各自的粒線體型蛋白質。我們實驗室研究發現non-AUG轉譯起始效率大約是AUG的三分之一或更少，而且周邊序列對non-AUG的轉譯效率影響也較對AUG大，除此之外，也發現最好的周邊序列是AAA (-3到-1核甘酸)。重複的non-AUG起始密碼(如ACGACG)通常能增加轉譯的效率；但某些重複的non-AUG起始密碼(如GUGGUG)會使周邊序列變差(尤其是-3核甘酸)，則其轉譯效率反而變差。另一個重大發現是：絕大部分酵母菌都只含有一個glycyl-tRNA synthetase (GlyRS)基因(稱做GRS1)，但是S. cerevisiae及V. polyspora卻有兩個相異的GlyRS基因(稱做GRS1及GRS2)。研究結果顯示：所有的酵母菌GRS1基因都是雙功能的，能同時做出細胞質及粒線體的GlyRS異構型，但是GRS2基因卻是非必需的。然而最令人驚奇的是：GRS2基因雖然在正常培養條件下不表現，它卻可以被一些逆境條件誘導表現，例如鹼性培養基(pH 8.0)或高溫生長條件(37°C)，且純化出來的GlyRS2蛋白質具有相當程度的胺醯化活性。在正常(30°C)及高溫條件下(37°C) GlyRS1與GlyRS2的穩定度都相當高，且在特定高溫條件下GRS2可以互補GRS1的剔除株，維持其正常生長。也許被誘導出來的GlyRS2在某些條件下可以取代GlyRS1的功能，另一種可能性則是GlyRS2參與其它代謝機制。

    Previous studies showed that ALA1 and GRS1 of Saccharomyces cerevisiae can initiate translation of their respective mitochondrial forms from a non-AUG codon. Our results showed that the translation efficiency of non-AUG initiation is about 30% (or less) relative to that of AUG initiation. In addition, it appeared that a non-AUG initiator codon is much more sensitive to its sequence context than is an AUG initiator codon, and AAA (the nucleotides at position -3 to -1 relative to the initiator) is the most favorable sequence context. Moreover, redundancy of non-AUG initiators, for instance ACGACG, significantly increased the translational efficiency. However, some redundant non-AUG initiators such as UUGUUG that have a poor sequence context (especially at position -3 relative to the second UUG codon), reduced the efficiency of translation. Another interesting discovery reported here was that the majority of yeast species possess a single glycyl-tRNA synthetase (GlyRS) gene (named GRS1). In contrast, S. cerevisiae and Vanderwaltozyma polyspora possessed two GlyRS genes (named GRS1 and GRS2). In all cases, GRS1 was dual-functional, because it encodes both cytoplasmic and mitochondrial forms of GlyRS. In contrast, GRS2 was pseudogene-like and dispensable for growth. Surprisingly, while GRS2 was silent under normal growth conditions (30°C), its expression was induced by certain stresses such as high temperature (37°C) and high external pH (pH 8.0). In addition, purified recombinant GlyRS2 retained a substantial level of aminoacylation activity. Both GlyRS1 and GlyRS2 were appreciably stable in vivo. When overexpressed, the GRS2 gene could rescue the growth defect of a GRS1 knockout strain. Altogether, these data suggest that GRS2 may function to substitute for GRS1 under certain circumstances. Alternatively, it may be involved in other as-yet-unidentified metabolic pathways.

    Table of contents 中文摘要 i Abstract ii 誌謝辭 iii Table of contents iV List of Figures Vi Overall introduction 1 Chapter I Translational efficiency of a non-AUG initiation codon is significantly affected by its sequence context in yeast 4 Abstract 5 Introduction 6 Experimental procedures 9 Results 12 Discussion 18 Chapter II Translational efficiency of redundant ACG initiator codons is enhanced by a favorable sequence context and remedial initiation 23 Abstract 24 Introduction 25 Experimental procedures 28 Results 32 Disscusion 40 Chapter III Vanderwaltozyma polyspora possesses a housekeeping and an inducible glycyl-tRNA synthetase gene 45 Abstract 46 Introduction 47 Experimental procedures 48 Results 52 Discussion 59 Summary 63 Reference 64 Figures 70 List of Figures Figure 1. The 5’-end sequence of GRS1. 70 Figure 2. Assay of protein expression patterns by Western blots. 71 Figure 3. Screening for important flanking sequences. 73 Figure 4. Effect of sequence context on the native TTG initiator. 75 Figure 5. Effect of sequence context on ATG and non-ATG initiators. 78 Figure 6. Context effect determined using lacZ as a reporter. 79 Figure 7. Efficiencies of translation initiation from various redundant and single non-AUG initiation codons. 82 Figure 8. Effect of the penultimate amino-terminal residue on the turnover of AlaRS-LexA fusions. 85 Figure 9. Efficiencies of translation initiation from various combinations of non-AUG initiation codons. 86 Figure 10. Effect of the nucleotide at position -3 on non-AUG initiation. 88 Figure 11. Efficiency of translation initiation from three successive ACG codons. 89 Figure 12. Efficiency of translation initiation from non-successive ACG codons. 91 Figure 13. Functional substitution of ATG1 of VAS1 with redundant GTG codons. 92 Figure 14. Comparison of yeast GlyRS1 and GlyRS2. 94 Figure 15. Cross-species complementation assays for yeast GlyRS genes. 95 Figure 16. Complementation assays for yeast GRS2 genes cloned into pADH. 97 Figure 17. Mapping the translation initiator codons of various yeast GlyRS genes. 100 Figure 18. Relative mRNA expression of GRS1 and GRS2 in Vanderwaltozyma polyspora and Saccharomyces cerevisiae. 102 Figure 19. Complementation assays for yeast GRS1 and GRS2 genes. 104 Figure 20. Protein stability assay for GlyRSs in vivo. 106 Figure 21. Aminoacylation assay for ScGlyRS1 and ScGlyRS2. 107 Figure 22. Aminoacylation assay for VpGlyRS1 and VpGlyRS2. 108 Figure 23. Aminoacylation assay at different temperatures. 109 Figure 24. Phylogenetic analysis of α2-dimeric GlyRS proteins. 111

    Reference
    1. Carter, C. W. Jr. (1993) Annu. Rev. Biochem. 62, 715-748
    2. Martinis, S. A., and Schimmel, P. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), 2nd Ed., pp. 887-901
    3. Giegé, R., Sissler, M., and Florentz, C. (1998) Nucleic Acids Res. 26, 5017-5035
    4. Pelchat, M., and Lapointe, J. (1999) Biochem. Cell. Biol. 77, 343-347
    5. Chatton, B., Walter, P., Ebel, J.-P., Lacroute, F., and Fasiolo, F. (1988) J. Biol. Chem. 263, 52-57
    6. Natsoulis, G., Hilger, F., and Fink, G. R. (1986) Cell 46, 235-243
    7. Turner, R. J., Lovato, M., and Schimmel, P. (2000) J. Biol. Chem. 275, 27681-27688
    8. Sherman, F., Stewart, J. W., and Schweingruber, A. M. (1980) Cell 20, 215-222
    9. Kozak, M. (1989) Mol. Cell. Biol. 9, 5073-5080
    10. Kozak, M. (1991) J. Biol. Chem. 266, 19867-19870
    11. Pisarev, A. V., Kolupaeva, V. G., Pisareva, V. P., Merrick, W. C., Hellen, C. U.T., and Pestova, T. V. (2006) Genes Dev. 20, 624-636
    12. Cigan, A. M., and Donahue, T. F. (1987) Gene 59, 1-18
    13. Baim, S. B., and Sherman, F. (1988) Mol. Cell. Biol. 8, 1591-1601
    14. Cigan, A. M., Pabich, E. K., and Donahue, T. F. (1988) Mol. Cell. Biol. 8, 2964-2975
    15. Zitomer, R. S., Walthall, D. A., Rymond, B. C., and Hollenberg, C. P. (1984) Mol. Cell. Biol. 4, 1191-1197
    16. Clements, J. M., Laz, T. M., and Sherman, F. (1988) Mol. Cell. Biol. 8, 4533-4536
    17. Chang, K. J., and Wang, C. C. (2004) J. Biol. Chem. 279, 13778-13785
    18. Tang, H. L., Yeh, L. S., Chen, N. K., Ripmaster, T., Schimmel, P., and Wang, C. C. (2004) J. Biol. Chem. 279, 49656-49663
    19. Abramczyk, D., Tchorzewski, M., and Grankowski, N. (2003) Yeast 20, 1045-1052
    20. Gazeau, M., Delort, F., Fromant, M., Dessen, P., Blanquet, S., and Plateau, P. (1992) J. Mol. Biol. 241, 378-389
    21. Leveque, F., Gazeau, M., Fromant, M., Blanquet, S., and Plateau, P. (1991) J. Bacteriol. 173, 7903-7910
    22. Bochner, B. R., Lee, P. C., Wilson, S. W., Cutler, C. W., and Ames, B. N. (1984) Cell 37, 225-232
    23. Bochner, B. R., Zylicz, M., and Georgopoulos, C. (1986) J. Bacteriol. 168, 931-935
    24. Fuge, E. K., and Farr, S. B. (1993) J. Bacteriol. 175, 2321-2326
    25. Brevet, A., Chen, J., Leveque, F., Plateau, P., and Blanquet, S. (1989) Proc. Natl. Acad. Sci. USA 89, 8275-8279
    26. Theoclitou, M. -E., E-Thaher, T. S. H., and Miller, A. D. (1994) J. Chem. Soc. Chem. Commun. 5, 659-661
    27. Dietrich, A., Weil, J. H., and Maréchal-Drouard, L (1992) Annu. Rev. Cell. Biol. 8, 115-131
    28. Wang, C. C., Chang, K. J., Tang, H. L., Hsieh, C. J., and Schimmel, P. (2003) Biochemistry 42, 1646-1651
    29. Souciet, G., Menand, B., Ovesna, J., Cosset, A., Dietrich, A., and Wintz, H. (1999) Eur. J. Biochem. 266, 848-854
    30. Huang, H. Y., Kuei, Y., Chao, H. Y., Chen, S. J., Yeh, L. S., and Wang, C. C. (2006) J. Biol. Chem. 281, 31430-31439
    31. Unbehaun, A., Borukhov, S. I., Hellen, C. U., and Pestova, T. V. (2004) Genes Dev. 18, 3078-3093
    32. Cheung, Y. N., Maag, D., Mitchell, S. F., Fekete, C. A., Algire, M. A., Takacs, J. E., Shirokikh, N., Pestova, T., Lorsch, J. R., and Hinnebusch, A. G. (2007) Genes Dev. 21, 1217-1230
    33. Kozak, M. (1990) Proc. Natl. Acad. Sci. USA 87, 8301-8305
    34. Chang, K. J., Lin, G., Men, L. C., and Wang, C. C. (2006) J. Biol. Chem. 281, 7775-7783
    35. Sikorski, R. S., and Hieter, P. (1989) Genetics, 122, 19-27
    36. Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 257, 3018-3025
    37. Kozak, M. (1999) Gene 234, 187-208
    38. Huang, H. Y., Tang, H. L., Chao, H. Y., Yeh, L. S., and Wang, C. C. (2006) Mol. Microbiol. 60, 189-198
    39. Slusher, L. B., Gillman, E. C., Martin, N. C., and Hopper, A. K. (1991) Proc. Natl. Acad. Sci. USA 88, 9789-9793
    40. Wolfe, C. L., Lou, Y. C., Hopper, A. K., and Martin, N. C. (1994) J. Biol. Chem. 269, 13361-13366
    41. Acland, P., Dixon, M., Peters, G., and Dickson, C. (1990) Nature 343, 662-665
    42. Saris, C. J., Domen, J., and Berns, A. (1991) EMBO J. 10, 655-664
    43. Hann, S. R., Sloan-Brown, K., and Spotts, G. D. (1992) Genes Dev. 6, 1229-1240
    44. Packham, G., Brimmell, M., and Cleveland, J. L. (1997) Biochem. J. 328, 807-813
    45. Riechmann, I. L., Ito, T., and Meyerowitz, E. M. (1999) Mol. Cell Biol. 19, 8505-8512
    46. Sadler, R., Wu, L., Forghani, B., Renne, R., Zhong, W., Herndier, B., and Ganem, D. (1999) J. Viol. 73, 5722-5730
    47. Yoon, H., and Donahue, T, F. (1992) Mol. Microbiol. 6, 1413-1419
    48. Huang, S., Elliott, R. C., Liu, P. S., Koduri, R. K., Weickmann, J. L., Lee, J. H., Blair, L. C., Ghosh-Dastidar, P., Bradshaw, R. A., Bryan, K. M., Einarson, B., Kendall, R. L., Kolacz, K. H., and Saito, K. (1987) Biochemistry 26, 8242-8246
    49. Polevoda, B., Norbeck, J., Takakura, H., Blomberg, A., and Sherman, F. (1999) EMBO J. 21, 6155-6168
    50. Chen, S. J., Lin, G., Chang, K. J., Yeh, L. S., and Wang, C. C. (2008) J. Biol. Chem. 283, 3173-3180
    51. Chen, S., Vetro, J. A., and Chang, Y. H. (2002) Arch. Biochem. Biophys. 398, 87-93
    52. Varshavsky, A. (1996) Proc. Natl. Acad. Sci. USA 93, 12142-12149
    53. Eriani, G., Delarue, M., Poch, O., Gangloff, J., and Moras, D. (1990) Nature 347, 203-206
    54. Schimmel, P. (1987) Annu. Rev. Biochem. 56, 125-158
    55. Ibba, M., Morgan, S., Curnow, A. W., Pridmore, D. R., Vothknecht, U. C., Gardner, W., Lin, W., Woese, C. R., and Söll, D. (1997) Science 278, 1119-1122
    56. Ostrem, D. L., and Berg, P. (1970) Proc. Natl. Acad. Sci. USA 67, 1967-1974
    57. Shiba, K., Schimmel, P., Motegi, H., and Noda, T. (1994) J. Biol. Chem. 269, 30049-30055
    58. Nada, S., Chang, P. K., and Dignam, J. D. (1993) J. Biol. Chem. 268, 7660-7667
    59. Kohli, J., Kwong, T., Altruda, F., Soll, D., and Wahl, G. (1979) J. Biol. Chem. 254, 1546-1551
    60. Chen, S. J., Ko, C. Y., Yen, C. W., and Wang, C. C. (2009) J. Biol. Chem. 284, 818-827
    61. Fersht, A. R., Ashford, J. S., Bruton, C. J., Jakes, R., Koch, G. L., and Hartley, B. S. (1975) Biochemistry 14, 1-4
    62. Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425
    63. Mirande, M. (1991) Prog. Nucleic Acid Res. Mol. Biol. 40, 95-142
    64. Wang, C. C., and Schimmel, P. (1999) J. Biol. Chem. 274, 16508-16512
    65. Wang, C. C., Morales, A. J., and Schimmel, P. (2000) J. Biol. Chem. 275, 17180-17186
    66. Chang, C. P., Lin, G., Chen, S. J., Chiu, W. C., Chen, W. H., and Wang, C. C. (2008) J. Biol. Chem. 283, 30699-30706
    67. Kaminska, M., Deniziak, M., Kerjan, P., Barciszewski, J., and Mirande, M. (2000) EMBO J. 19, 6908-6917
    68. Kaminska, M., Shalak, V., and Mirande, M. (2001) Biochemistry 40, 14309-14316
    69. Francin, M., Kaminska, M., Kerjan, P., and Mirande, M. (2002) J. Biol. Chem. 277, 1762-1769
    70. Francin, M., and Mirande, M. (2006) Biochemistry 45, 10153-10160
    71. Simos, G., Segref, A., Fasiolo, F., Hellmuth, K., Shevchenko, A., Mann, M., and Hurt, E. C. (1996) EMBO J. 15, 5437-5448
    72. Godinic, V., Mocibob, M., Rocak, S., Ibba, M., and Weygand-Durasevic, I. (2007) FEBS. J. 274, 2788-2799
    73. Clark, R. L., and Neidhardt, F. C. (1990) J. Bacteriol. 172, 3237-3243
    74. Kawakami, K., Ito, K., and Nakamura, Y. (1992) Mol. Microbiol. 6, 1739-1745
    75. Guo, R. T., Chong, Y. E., Guo, M., and Yang, X. L. (2009) J. Biol. Chem. 284, 28968-28976
    76. Varshavsky, A. (1983) Cell 34, 711-712
    77. Maréchal-Drouard, L., Small, I., Weil, J. H., and Dietrich, A. (1995) Meth. Enzymol. 260, 310-327
    78. Duchene, A. M., Peters, N., Dietrich, A., Cosset, A., Small, I., and Wintz, H. (2001) J. Biol. Chem. 276, 15275-15283
    79. Mazauric, M. H., Reinbolt, J., Lorber, B., Ebel, C., Keith, G., Giegé, R., and Kern, D. (1996) Eur. J. Biochem. 241, 814-826
    80. Logan, D. T., Mazauric, M. H., Kern, D., and Moras, D. (1995) EMBO J. 14, 4156-4167
    81. Chiu, W. C., Chang, C. P., Wen, W. L., Wang, S. W., and Wang, C. C. (2010) Mol. Biol. Evol. 27, 1415-1424
    82. Chiu, W. C., Chang, C. P., and Wang, C. C. (2009) J. Biol. Chem. 284, 23954-23960
    83. Brown, J. R., and Doolittle, W. F. (1997) Microbiol. Mol. Biol. Rev. 61, 456-502
    84. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680

    QR CODE
    :::