酵母菌HTS1基因轉譯起始機制之研究

Translation initiation of HTS1 in yeast

郭亦亦(Yi-Yi Kuo)

指導教授 ： 王健家

國立中央大學/理學院/生命科學系/碩士(2008年)

摘要

在酵母菌中有兩套不同的細胞核基因，分別控制細胞質及粒腺體 aminoacyl-tRNA synthetase（aaRS）的合成。這兩套細胞核基因在轉譯完成後，其中一個 aaRS 會留在細胞中進行細胞質蛋白質合成，而另外一個帶有粒腺體標的訊號的 aaRS 則會被送到粒腺體中去作用。然而Saccharomyces cerevisiae HisRS（ScHisRS）只用一個HTS1基因轉譯出兩種異構型酵素，分別在粒腺體與細胞質中進行胺醯化反應。HTS1基因上含有兩個in-frame的ATG，分別為ATG1及ATG21。根據前人實驗的結果發現HTS1基因，會轉錄出兩種mRNA，較長的mRNA包含了AUG1及AUG21，而較短的mRNA只有AUG21。在本實驗中我們選殖了Schizosaccharomyces pombe、Candida albicans和Aspergillus fumigatus的HTS1基因，利用互補性實驗鑑定這些基因的轉譯起始密碼，結果發現它們也有兩個in-frame的AUG，分別做出粒腺體與細胞質酵素。此外當我們比較ScHisRS及大腸桿菌的HisRS序列後，其中ScHisRS較大腸桿菌的N端多了一段34個胺基酸的附加區段，此附加區段並不能活化大腸桿菌GlnRS，因此推測ScHisRS附加區段不能增加E. coli GlnRS對酵母菌tRNAHis結合的親和力。此外，刪除了ScHisRS附加區段對於其細胞質和粒腺體的活性是沒有影響的，所以我們推測在in vivo的情況下HisRS的附加區段對於HisRS的催化活性並非必需的。

關鍵字

轉譯起始機制 ； 酵母菌

並列摘要

In yeast, there are two sets of aminoacyl-tRNA synthetases, one localized in the cytoplasm and the other in the mitochondria. Most of the mitochondrial tRNA synthetases are encoded by nuclear genes distinct from those encoding their cytoplasmic counterparts. However, some mitochondrial tRNA synthetases are encoded by the same genes that code for their cytoplasmic homologues. For example, the cytoplasmic and mitochondrial histidyl-tRNA synthetases of Saccharomyces cerevisiae (ScHisRS) are encoded by the same nuclear gene, HTS1, through alternative initiation of translation from two in-frame AUG codons. The gene specifies two messages, the longer one with two 5’-end in-frame AUGs and the short one with only the second AUG. In this study, we showed that the HisRS genes of Schizosaccharomyces pombe、Candida albicans and Aspergillus fumigatus have only one gene that also contain two in-frame AUG initiation codons. The mitochodrial and cytosolic forms are translated from the first and second AUG initiation codons, respectively. Besides, we found that ScHisRS had a lysine-rich N-terminal polypeptide extension of 34 residues, which was absent from its E. coli counterpart. Attachment of this appended domain to E. coli glutaminyl-tRNA synthetase did not enhance the enzyme’s tRNA-binding and aminoacylation activity toward yeast tRNAs. Deletion of the appended domain of ScHisRS had little effect on the enzyme’s complementation activity in vivo. These results suggest that the appended domain is not essential for the cytoplasmic and mitochondrial functions of ScHisRS in vivo.

並列關鍵字

translation initiation ； Histidyl-tRNA synthetase

參考文獻

Wang, C. C., Chang, K. J., Tang, H. L., Hsieh, C. J., Schimmel, P. (2003) Mitochondrial form

Arnez, J. G., Harris, D.C., Mitshler, A., Rees, B., Frnacklyn, C.S., and Moras, D. (1995) Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenelate. EMBO J 14: 4143.

Arnez, J. G. and Moras, D. (1997) Structural and functional considerations of the aminoacylation reaction. Trends Biochem. Sci 22: 211-216.

Carter, C. W. Jr. (1993) Cognition, mechanism, and evolutionary relationships in

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.