探討酵母菌 Histidine tRNA 之辨識

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：108

、訪客IP：18.119.122.106

姓名

李宜學(Yi-Siao Li) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討酵母菌 Histidine tRNA 之辨識

相關論文

★ Kineosphaera limosa 菌株中 phaC 基因之序列分析	★ 剪力和組織蛋白去乙醯酶在動靜脈廔管失效扮演的角色
★ Classification of powdery mildews on ornamental plants in northern Taiwan	★ 秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla
★ Bacillus thuringiensis contains two prolyl-tRNA synthetases of different origins	★ Recognition of tRNA His isoacceptors by human HisRS isoforms
★ Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P	★ Functional characterization of a noncanonical ProRS in Toxoplasma gondii
★ tRNA aminoacylation by a naturally occurring mini-AlaRS	★ Functional Repurposing of C-Ala Domains
★ Recognition of a non-canonical tRNAAla by a non-canonical alanyl-tRNA synthetase	★ 探討Alanyl-tRNA synthetase的演化及專一性
★ 酵母菌valyl-tRNA synthetase附加區段的生物功能之探討	★ 探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能
★ 探討酵母菌Valyl-tRNA synthetase的生化活性	★ 酵母菌轉譯起始機制的研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

在酵母菌體內具有兩套aminoacyl-tRNA synthetases (aaRSs)，一套作用在細胞質，另一套作用在粒線體。然而細胞質和粒線體的HisRS卻是由同一個基因HTS1經由兩個不同的AUG轉譯起始點做出來的。過去的研究指出大腸桿菌和酵母菌的tRNAHis 在-1:73位置是重要的aaRS辨識核苷酸，而在酵母菌細胞質和粒線體tRNAHis相對位置分別是G-1:A73和G-1:C73，在大腸桿菌中則是G-1:C73，且大腸桿菌的histidyl-tRNA synthetase (HisRS) 無法取代酵母菌細胞質HisRS的功能，若將其送入粒線體則可取代。我們也進一步在體外實驗中證實，大腸桿菌HisRS無法辨識G-1:A73的酵母菌細胞質tRNAHis。由此推論G-1:A73和G-1:C73分別是真核生物細胞質和粒線體的保守辨識位置，我們的實驗結果也顯示人類細胞含有HisRS分為粒線體與細胞質異構型HisRS，它們可以分別取代酵母菌粒線體和細胞質的HisRS功能。值得一提的，我們的實驗結果發現果蠅和線蟲的HisRS也都是雙重功能基因，可以同時作用在細胞質和粒線體，且果蠅HisRS應該是由非AUG起始密碼做出帶有粒線體標的訊號的異構型。除此之外，人類和酵母菌HisRS經過結構域互換的結果顯示對HisRS 而言N端結構域才是辨識tRNAHis 的重要區域。因此我們更細部探討，發現在體內實驗中酵母菌HisRS的motif 2 loop突變會影響它在粒線體中的活性，但不影響在細胞質。由體外胺醯化反應結果可以發現motif 2 loop突變使其催化細胞質tRNAHis效率約下降30倍，而且幾乎無法辨識粒線體tRNAHis。由此發現我們認為酵母菌HisRS之motif 2 loop對於辨識-1:73位置相當重要，尤其是G-1:C73。這對於雙功能HisRS基因在演化上的策略提供了新的線索。

摘要(英)

There are two distinct sets of aminoacyl-tRNA synthetases (aaRSs) in yeast, one localized in the cytoplasm and the other in mitochondria. Paradoxically, there is only one histidyl-tRNA synthetase (HisRS) gene, HTS1, in the yeast nuclear genome. Recent studies indicate that this gene encodes both cytoplasmic and mitochondrial forms of HisRS through alternative initiation of translation from two in-frame AUG initiator codons. We report herein that a similar, dual-functional phenotype is conserved in the HTS1 genes of many eukaryotes. While E. coli tRNAHis contains a G-1:C73 base pair as the major determinant, yeast cytosolic and mitochondrial tRNAHis isoacceptors respectively contain G-1:A73 and G-1:C73 at the same positions. We found that human cytoplasmic and mitochondrial HisRS can respectively substitute for the cytoplasmic and mitochondrial activities of yeast HTS1. Furthermore, The HisRS gene of Drosophila melanogaster and Caenorhabditis elegans are also dual functional in yeast. Domain swapping between yeast HisRS and human HisRS suggested that the N-terminal domains is important for tRNAHis recognition. Moreover, mutations in motif 2 loop largely impaired the mitochondrial but slightly cytoplasmic activity. This finding suggests that the motif 2 loop of yeast HisRS is important for recognition of G-1:C73. This may provide a clue of evolution strategy of the dual-functional HTS1 gene.

關鍵字(中)

★ 酵母菌
★ 胺醯-tRNA合成酶

關鍵字(英)

★ aaRS
★ tRNAHis
★ G-1
★ yeast

論文目次

目錄
中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 VIII
縮寫檢索表 IX
第一章緒論 1
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 1
1.1.1 aaRS的功能 1
1.1.2 aaRSs 的分類 2
1.2 原核生物與真核生物蛋白質轉譯方式 2
1.2.1原核生物與真核生物蛋白質合成 2
1.2.2 真核細胞aaRS雙重功能基因 3
1.3 Histidyl-tRNA synthetase (HisRS)的簡介 4
1.3.1 HisRS的生化特性 4
1.3.2酵母菌的HTS1基因 4
1.4 tRNAHis的辨認位置 5
1.4.1 tRNAHis特別且保守的G-1核苷酸 5
1.4.2 真核和原核生物tRNAHis的N73鹼基 6
第二章材料與方法 7
2.1 菌株、載體及培養基 7
2.2 大腸桿菌勝任細胞的製備與轉型作用 8
2.2.1大腸桿菌勝任細胞的製備 8
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) 9
2.3 酵母菌勝任細胞的製備與轉型作用 9
2.3.1酵母菌勝任細胞的製備 9
2.3.2酵母菌勝任細胞的轉型作用 10
2.4 質體之選殖 10
2.5 點突變 (Site-directed Mutagenesis) 11
2.6 功能性互補試驗 (Complementation)―測試細胞質功能 12
2.7 功能性互補試驗 (Complementation)―測試粒線體功能 13
2.8 蛋白質製備 (Protein preparation) 14
2.9 SDS-PAGE之蛋白質分子量分析 15
2.10 西方點墨法 (Western Blotting) 15
2.11 酵母菌融合蛋白質的表現和純化 17
2.12 aaRS胺醯化反應 (aminoacylation assay) 20
第三章結果 22
3.1 酵母菌HisRS 的生化活性 22
3.2 酵母菌tRNAHis 其它識別元素 23
3.3 高等真核生物HisRS專一性與功能 24
3.4 G-1對真核生物HisRS的重要性 26
3.5 酵母菌和人類HisRS 之N端與C端結構域功能 27
3.6 酵母菌HisRS Motif 2 loop的重要性 28
第四章討論 29
參考文獻 33
圖表 36

參考文獻

Burbaum JJ, Schimmel P (1991) Assembly of a class I tRNA synthetase from products of an artificially split gene. Biochemistry 30: 319-324
Burbaum JJ, Schimmel P (1992) Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence. Protein Sci 1: 575-581
Carter CW, Jr. (1993) Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu Rev Biochem 62: 715-748
Cavadini P, Gakh O, Isaya G (2002) Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase. Methods 26: 298-306
Chang KJ, Wang CC (2004) Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. J Biol Chem 279: 13778-13785
Chiu MI, Mason TL, Fink GR (1992) HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation. Genetics 132: 987-1001
Freist W, Verhey JF, Ruhlmann A, Gauss DH, Arnez JG (1999) Histidyl-tRNA synthetase. Biol Chem 380: 623-646
Giege R, Sissler M, Florentz C (1998) Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res 26: 5017-5035
Gu W, Jackman JE, Lohan AJ, Gray MW, Phizicky EM (2003) tRNAHis maturation: an essential yeast protein catalyzes addition of a guanine nucleotide to the 5’ end of tRNAHis. Genes Dev 17: 2889-2901
Hawko SA, Francklyn CS (2001) Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element. Biochemistry 40: 1930-1936
Heinemann IU, Nakamura A, O’Donoghue P, Eiler D, Soll D (2012) tRNAHis-guanylyltransferase establishes tRNAHis identity. Nucleic Acids Res 40: 333-344
Himeno H, Hasegawa T, Ueda T, Watanabe K, Miura K, Shimizu M (1989) Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation. Nucleic Acids Res 17: 7855-7863
Martinis SA, Plateau P, Cavarelli J, Florentz C (1999) Aminoacyl-tRNA synthetases: a family of expanding functions. Mittelwihr, France, October 10-15, 1999. EMBO J 18: 4591-4596
Martinis SA, Schimmel P (1993) Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity. J Biol Chem 268: 6069-6072
McClain WH (1993) Rules that govern tRNA identity in protein synthesis. J Mol Biol 234: 257-280
Mireau H, Lancelin D, Small ID (1996) The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases. Plant Cell 8: 1027-1039
Morl M, Marchfelder A (2001) The final cut. The importance of tRNA 3’-processing. EMBO Rep 2: 17-20
Natsoulis G, Hilger F, Fink GR (1986) The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell 46: 235-243
Paschen SA, Neupert W (2001) Protein import into mitochondria. IUBMB Life 52: 101-112
Preston MA, Phizicky EM (2010) The requirement for the highly conserved G-1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase. RNA 16: 1068-1077
Ribas de Pouplana L, Schimmel P (2001) Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stem. Cell 104: 191-193
Ripmaster TL, Shiba K, 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 U S A 92: 4932-4936
Rosen AE, Brooks BS, Guth E, Francklyn CS, Musier-Forsyth K (2006) Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs. RNA 12: 1315-1322
Schimmel P (1987) Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs. Annu Rev Biochem 56: 125-158
Sikorski RS, 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
Souciet G, Menand B, Ovesna J, Cosset A, Dietrich A, 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
Sprinzl M, Vassilenko KS (2005) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res 33: D139-140
Tang HL, Yeh LS, Chen NK, Ripmaster T, Schimmel P, Wang CC (2004) Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J Biol Chem 279: 49656-49663
Wolstenholme DR, Macfarlane JL, Okimoto R, Clary DO, Wahleithner JA (1987) Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms. Proc Natl Acad Sci U S A 84: 1324-1328
Yan W, Francklyn C (1994) Cytosine 73 is a discriminator nucleotide in vivo for histidyl-tRNA in Escherichia coli. J Biol Chem 269: 10022-10027
Yuan J, Gogakos T, Babina AM, Soll D, Randau L (2011) Change of tRNA identity leads to a divergent orthogonal histidyl-tRNA synthetase/tRNAHis pair. Nucleic Acids Res 39: 2286-2293

指導教授

王健家(Chien-Chia Wang)

審核日期

2013-7-25

推文