Title page for 93224007


[Back to Results | New Search]

Student Number 93224007
Author Yu Kuei(桂妤)
Author's Email Address No Public.
Statistics This thesis had been viewed 1793 times. Download 568 times.
Department Life Science
Year 2005
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Study of a bifunctional yeast tRNA synthetase of Candida albicans
Date of Defense 2006-07-07
Page Count 80
Keyword
  • alanyl-tRNA synthetase
  • bifunctional
  • Candida albicans
  • Abstract It was previously shown that the cytoplasmic and mitochondrial activities of alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two translational products of ALA1, one initiates at the AUG codon closest to the 5’-end of its mRNA transcripts and the other at upstream in-frame redundant non-AUG codons (i. e., ACG(-25) and ACG(-24)). In this thesis, we report the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae; however, several points regarding the pattern of gene expression differ from mechanisms used to regulate expression of ScALA1. First, the results of 5’-RACE showed expression of only a single transcript whose 5’-end mapped to nucleotide position -24 relative to ATG1. However, this single transcript codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8 codons apart. Complementation assays showed that the AUG1-initiated protein can complement for both cytoplasmic and mitochondrial activities of ScALA1, while the AUG9-initiated protein functions only in the cytoplasm. Fractionation assays also showed that the AUG1-initiated protein form can be partitioned in both compartments; however, the AUG9-initiated protein form was exclusively confined to the cytoplasm. Next, we tested whether the mechanism used by CaALA1 to initiate translation was due to “leaky scanning”. Therefore, a series of point mutations were introduced into the sequence context surrounding the first initiator. Results showed that the efficiencies of translation initiation by the two initiators could be influenced by changing the sequence context of AUG1, consistent with the leaky scanning model of translation initiation. To our knowledge, this appears to be the first example in yeast wherein a naturally occurring form of a tRNA synthetase can play roles in both compartments.
    Table of Content 中文摘要I
    英文摘要II
    誌謝III
    目錄IV
    圖表目錄VI
    縮寫檢索表VII
    第一章 緒論1
    1. Aminoacyl-tRNA synthetase (aaRS) 的簡介1
    2. 原核與真核細胞在轉譯方式上的差異1
    3. 少數的真核細胞 aaRS 只有一個細胞核基因2
    4. 比較 S. cerevisiae 與 Candida albicans 之間 AlaRS 蛋白質表現差異4
    5. 真核細胞的轉譯機制4
    6. 研究目的5
    第二章 材料與方法6
    1. 使用之菌株、載體及培養基6
    2. 大腸桿菌勝任細胞的製備與轉型作用6
    3. 酵母菌勝任細胞的製備與轉型作用8
    4. 質體之選殖9
    5. 鑑定 CaALA1 mRNA 的 5’端10
    6. 點突變 (Site-directed Mutagenesis) 13
    7. 功能性互補試驗 (Complementation ) —測試細胞質功能14
    8. 功能性互補試驗 (Complementation ) —測試粒線體功能15
    9. 蛋白質製備 (Protein Preparation)17
      10. SDS-PAGE之蛋白質分子量分析18
    11. 西方氏點墨法 (Western blotting)19
    12. 酵母菌粒線體的分離 (Enrichment of mitochondria)20
    第三章 結果22
    1. CaALA1 基因序列的分析22
    2. CaALA1 具有細胞質與粒線體雙重功能 23
    3. CaALA1 可由不同的轉譯起始點合成兩個蛋白質異構型24
    4. CaAlaRS 在細胞中的分布情形25
    5. 比較 AUG1 與 AUG9 的轉譯起始效率28
    6. 以回報基因分析 CaAlaRS 的合成方式28
    7. 週邊序列影響轉譯起始點的效率29
    第四章 討論 32
    1. CaALA1 為一個雙重功能基因32
    2. 一個 CaAlaRS 異構型可以同時作用於兩個胞器33
    3. 粒線體基質胜??切割效率的探討34
    4. 酵母菌 Leaky scanning 的探討35
    第五章 參考文獻37
    圖表41
    附錄58
    Reference Abramczyk, D., Tchorzewski, M., Grankowski, N. (2003) Non-AUG translation initiation of mRNA encoding acidic ribosomal P2A protein in Candida albicans. Yeast 12: 1045-1052
    Burbaum, J. J., Schimmel, P. (1991) Structural relationships and the classification of aminoacyl-tRNA synthetases. J. Biol. Chem. 266:16965-8.
    Carter, C. W. Jr. (1993) Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu. Rev. Biochem. 62: 715-748
    Chang, K. J., and Wang, C. C. (2004) Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. J. Biol. Chem. 279: 13778-13785
    Chatton, B., Walter, P., Ebel, J. P., Lacroute, F., and Fasiolo, F. (1988) The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. J. Biol. Chem. 263: 52-57.
    Chiu, M. I., Mason, T. L., Fink, G. R. (1992) HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation. Genetics. 132: 987-1001
    Cigan, A. M., Donahue, T. F. (1987) Sequence and structural features associated with  translational initiator regions in yeast. Gene 59(1): 1-18.
    Daum, G., Bohni, P. C., Schatz, G. (1982) Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem. 257:13028-33.
    Dircks, L. K., Poyton, R. O. (1990) Overexpression of a leaderless form of yeast cytochrome c oxidase subunit Va circumvents the requirement for a leader peptide in mitochondrial import. Mol Cell Biol. 10:4984-6.
    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.
    Gakh, O., Cavadini, P., Isaya, G. (2002) Mitochondrial processing peptidases. Biochim Biophys Acta. 1592: 63-77.
    Gieg?, R., Sissler, M., and Florentz, C. (1998) Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26: 5017-5035.
    Kozak, M. (1989) Context effects and inefficient initiation at non-AUG codons in eukaryotic cell-free translation systems. Mol. Cell. Biol. 9: 5073-5080.
    Kozak, M. (1990) Downstream secondary structure facilitates recognition of initiator  codons by eukaryotic ribosomes. Proc. Natl. Acad. Sci. USA 87: 8301-8305
    Kozak, M. (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266: 19867-19870.
    Kozak, M. (1997) Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J. 16: 2482-92
    Kozak, M. (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234: 187-208
    Mar?chal-Drouard, L., Weil, J. H., and Dietrich, A. (1993) Transfer RNAs and transfer RNA genes in plants. Annu. Rev. Cell. Biol. 8: 115-131.
    Martinis, S. A., Schimmel, P. (1993) Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity. J. Biol. Chem. 268: 6069-72.
    Mirande, M. (1991) Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol. 40:95-142.
    Mireau, H., Lancelin, D., and Small, I. D. (1996) The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases. Plant Cell 8: 1027-1039
    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
    Natsoulis, G., Hilger, F., and Fink, G. R. (1986) The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell 46: 235-243.
    Neupert, W. (1997) Protein import into mitochondria. Annu Rev Biochem. 66: 863-917.
    Peeters, N., and Small, I. (2001) Dual targeting to mitochondria and chloroplasts. Biochim. Biophys. Acta 1541: 54–63
    Ribas de Pouplana, L., Turner, R. J., Steer, B. A., Schimmel, P. (1998) Genetic code origins: tRNAs older than their synthetases? Proc Natl Acad Sci U S A. 95: 11295-300.
    Ripmaster, T. L., Shiba, K., and Schimmel, P. (1995) Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc. Natl. Acad. Sci. USA 92: 4932-4936
    Sass, E., Blachinsky, E., Karniely, S., and Pines, O. (2001) Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini. J. Biol. Chem. 276: 46111-46117
    Sass, E., Karniely, S., and Pines, O. (2003) Folding of fumarase during mitochondrial import determines its dual targeting in yeast. J. Biol. Chem. 278: 45109-45116 
    Schimmel, P., R. and Soll, D. (1979) Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem. 48:601-48.
    Sikorski, R. S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27
    Slusher, L. B., Gillman, E. C., Martin, N. C., and Hopper, A. K. (1991) mRNA leader length and initiation codon context determine alternative AUG selection for the yeast gene MOD5. Proc. Natl. Acad. Sci. USA 88: 9789-9793
    Souciet, G., Menand, B., Ovesna, J., Cosset, A., Dietrich, A., and Wintz, H. (1999) Characterization of two bifunctional Arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl-tRNA synthetase and threonyl-tRNA synthetase by alternative use of two in-frame AUGs. Eur. J. Biochem. 266: 848-854.
    Strobel, G., Zollner, A., Angermayr, M., and Bandlow, W. (2002) Competition of Spontaneous Protein Folding and Mitochondrial Import Causes Dual Subcellular Location of Major Adenylate Kinase. Mol. Biol. Cell 13: 1439–1448
    Tang, H. L., Yeh, L. S., Chen, N. K., Ripmaster, T., Schimmel, P., Wang, C. C. (2004) Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J. Biol. Chem. 279: 49656-63
    Turner, R. J., Lovato, M., Schimmel, P. (2000) One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions. J. Biol. Chem. 275: 7681-8.
    Tzagoloff, A., Vambutas, A., and Akai, A. (1989) Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase. Eur. J. Biochem. 179: 365–371.
    Tzagoloff, A., Gatti, D., Gampel, A. (1990) Mitochondrial aminoacyl-tRNA synthetases. Prog Nucleic Acid Res Mol Biol. 39:129-58.
    Wang, C. C., Chang, K. J., Tang, H. L., Hsieh, C. J., Schimmel, P. (2003) Mitochondrial form of a tRNA synthetase can be made bifunctional by manipulating its leader peptide. Biochemistry 42: 1646-51.
    Wolfe, C. L., Lou, Y. C., Hopper, A. K., Martin, N. C. (1994) Interplay of heterogeneous transcriptional start sites and translational selection of AUGs dictate the production of mitochondrial and cytosolic/nuclear tRNA nucleotidyltransferase from the same gene in yeast. J. Biol. Chem.269: 13361-6.
    黃曉芸 (2005) 酵母菌 ALA1 基因轉譯起始機制的研究。中央大學碩士論文
    Advisor
  • Wang Chien-Chia(王健家)
  • Files
  • 93224007.pdf
  • approve in 1 year
    Date of Submission 2006-07-18

    [Back to Results | New Search]


    Browse | Search All Available ETDs

    If you have dissertation-related questions, please contact with the NCU library extension service section.
    Our service phone is (03)422-7151 Ext. 57407,E-mail is also welcomed.