先前研究已知，在Saccharomyces cerevisiae 中，粒腺體以及細胞質功
能的alanyl-tRNA synthetase (稱為AlaRS)是由ALA1 基因所提供。ALA1 基
因利用最靠近mRNA 5’ 端的ATG (1)轉譯出細胞質所需的AlaRS，再利用
ATG1 上游的二個重複ACG (即ACG (-25)和ACG (-24)) 做出粒腺體異構
型，主要仍以ACG (-25)為主。本篇論文著重於酵母菌中使用non-ATG 作為
轉譯起始密碼相關機制的研究。我們以ALA1 為對象去篩選在自然的情況
下，是否有其它的密碼可以取代原本提供粒腺體功能的轉譯起始密碼
(ACG (-25))，篩選結果發現和高等真核生物一樣，酵母菌可以使用和ATG
只差一個核苷酸的密碼 (ATA, ATU, ATC, GTG, TTG, CTG)作為轉譯起始
點，除此之外，我們也篩選到非常罕見且一般不被認為的轉譯起始點密碼
(AGC, CGC, CAT, CAC)可以取代ACG (-25)。我們利用西方點墨法進一步定
量發現，周圍序列-3 ~ -1 的核苷酸對轉譯效率有非常大的影響；另外我們
發現ACG (-25)下游的一個二級結構對轉譯效率也有非常大的影響，其詳細
機制有待進一步研究。
論文第二部份，著重於酵母菌中valyl-tRNA synthetase (ValRS)附加區段
的研究。我們發現這個附加區段具有非專一性tRNA 鍵結的能力，幫助tRNA
鍵結至valine 的酵素上 (Kd 約為2 μM)，而對此附加區段做部分刪除，例
如Δ32-71，便破壞酵素對tRNA 鍵結的能力及酵素胺醯化的活性。除此之
外，先前研究研究顯示附加區段活化轉錄的活性，而我們發現ValRS 可藉
由附加區段將GFP 送入細胞核中，這些結果顯示ValRS 可能參與細胞核基
因的調控。

Recent studies in Saccharomyces cerevisiae have shown that ALA1 (coding
for alanyl-tRNA synthetase) initiates the translation of its mitochondrial isoform
from two consecutive ACG triplets, with the first ACG being the more robust. In
the work described here we focuse on the mechanism of non-ATG initiation in
yeast. To explore if any other non-ATG triplets can serve a similar function, the
first and second ACG triplets were replaced by a random triplet and ACC,
respectively, and the resultant constructs were screened for candidates
expressing the mitochondrial activity. We report here that, in addition to ATG,
six common non-ATG initiators (ATA, ATU, ATC, GTG, UTG, and CTG) and
four rare non-ATG initiators (AGC, CGC, CAT, and CAC) can each functionally
substitute for the ACG initiators. Western blot analysis suggests that initiating
activity of the non-ATG initiators is significantly affected by the nucleotides at
their relative positions -3 ~ -1. Furthermore, a putative pseudoknot structure
17-nucleotide downstream of the non-AUG initiation site also appears to be
important for the initiation activity.
A second part of the thesis is focused on the valyl-tRNA synthetase (ValRS)
appended domain (ad) of Saccharomyces cerevisiae. We show here that this
domain is a non-specific tRNA-binding domain (Kd ~ 2 μM) that contributes
significantly to the tRNA-binding activity of the valine enzyme. Thus, even a
small deletion in the appended domain, such as Δ32-71, has a devastating effect
on the enzyme’s tRNA-binding and aminoacylation activities. In addition we
show here that the ad can deliver a green fluorescent protein in to the nucleus,
further reinforcing the notion that ValRS could be involved regulation of nuclear
gene.

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
Arnez, J. G. and Moras, D. (1997) Structural and functional considerations of the aminoacylation reaction. Trends Biochem. Sci. 22:211-216.
Beuning, P. J. and Musier-Forsyth, K. (2000) Hydrolytic editing by a classⅡ-tRMA synthetases. PNAS 97: 8916-8920.
Binns, N. and Masters, M. (2002) Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU. Mol Microbiol. 44:1287-98.
Burbaum, J. J., Schimmel, P. (1991) Structural relationships and the classification of aminoacyl-tRNA synthetases. J Biol Chem. 266:16965-8.
Butler, J. S., Springer, M., Dondon, J., Graffe, M., Grunberg-Manago, M. (1986) Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo. J Mol Biol. 192:767-80.
Chatton, B., Walter, P., Ebel, J. P., Lacroute, F., Fasiolo, F. (1988) The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. J Biol Chem. 263:52-7.
Cigan, A. M., Pabich, E. K., and Donahue, T. F. (1988) Mutational analysis of the His4 translation initiator region in Saccharomyces cerevisiae. Mol. Cell. Biol. 8: 2964-2975.
Clements, J. M., Laz, T. M., and Sherman, F.(1988) Efficiency of translation initiation by non-AUG codon in Saccharomyces cerevisiae. Mol. Cell. Biol. 8: 4533-4536.
Cahuzac, B., Berthonneau, E., Birlirakis, N., Guittet, E. and Mirande, M. (2000) A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases. EMBO J. 19: 445-52.
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.
Donahue, T. F., Cigan, A. M. (1988) Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region. Mol Cell Biol. 8:2955-2963.
Farrow, M. A.,Nordin, B. E. and Schimmel, P.(1999) Nucleotide determinants for tRNA-dependent amino acid discrimination by a classⅠtRNA synthetase.Biochemistry.38:16898-16903.
Grill, S., Gualerzi, C. O., Londei, P. and Blasi, U. (2000) Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation. EMBO J. 19:4101-10.
Gold, L. (1988) Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 57:199-233.
Hartz, D., McPheeters, D. S., Green, L. and Gold L. (1991) Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA. J Mol Biol. 218:99-105
Hendrickson, T. L., Nomanbhoy, T. K. and Schimmel, P. (2000) Errors from selective disruption of the editing center in a tRNA synthetase. Biochemistry.39: 108-8186.
Heck, J. D, and Hatfield, G. W. (1988) Valyl-tRNA synthetase gene of Escherichia coli K12. Molecular genetic characterization. J Biol Chem. 263:857-67.
Jordana, X., Chatton, B., Paz-Weisshaar, M., Buhler, JM., Cramer, F., Ebel, JP., Fasiolo, F. (1987) Structure of the yeast valyl-tRNA synthetase gene (VASI) and the homology of its translated amino acid sequence with Escherichia coli isoleucyl-tRNA synthetase. J Biol Chem. 262: 189-94.
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.
Ludmerer, S. W., Wright, D. J., Schimmel, P. (1993) Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain. J Biol Chem.268:5519-23.
Lin, L., Hale, S. P. and Schimmel, P. (1996) Aminoacylation error correction. Nature.384:33-44.
Lin, L., and Schimmel, P. (1996) Mutational analysis suggests the same design for editing activites of two tRNA aynthetase.Biochemistry.35: 5596-5601.
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. The Plant Cell 8: 1027-1039
Putney, S. D.and Schimmel, P. (1981) An aminoacyl tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription. Nature. 291:632-5.
Ribas de Pouplana, L. and Schimmel, P. (2001) Two classes of tRNA synthetases suggested by sterically Compatible dockings on tRNA acceptor stem. Cell 104: 191-193.
Ringquist, S., Shinedling, S., Barrick, D., Green, L., Binkley, J., Stormo, G.D. and Gold, L. (1992) Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol. 6:1219-29.
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
Romby, P., Wakao, H., Westhof, E., Grunberg-Manago, M., Ehresmann, B., Ehresmann, C. and Ebel, J.P. (1990) The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex.
Biochim Biophys Acta. 1050:84-92.
Sacerdot, C., Chiaruttini, C., Engst, K., Graffe, M., Milet, M., Mathy, N., Dondon, J.and Springer., M. (1996) The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli. Mol Microbiol. 21:331-46.
Shine, J., Dalgarno, L. (1974) The 3''-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 71:1342-6.
Sherman, F., Stewart, J.W., Schweingruber, A.M., (1980) Mutants of yeast initiating translation of iso-1-cytochrome c within a region spanning 37 nucleotides. Cell.20:215-222.
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
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.
Schimmel, P., R. and Soll, D. (1979) Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem. 48:601-48.
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.
Vellanoweth, R. L.and Rabinowitz, J. C. (1992) The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol Microbiol. 6:1105-14.
Wang, C. C. and Schimmel, P. (1999) Species barrier to RNA recognition overcome with nonspecific RNA binding domains. J. Biol. Chem 274: 16508-16512.
謝佳容 ( 2002) 酵母菌valyl-tRNA synthetase附加區段的生物功能之探討。中央大學碩士論文
葉蟬嫻 ( 2003) 探討酵母菌ALA1基因的 non-AUG轉譯機制中央大學碩士論文
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
林明琁 ( 2005) 探討一個真核tRNA合成酶的附加區段之轉錄活化活性。中央大學碩士論文