人類細胞質AlaRS的C-Ala在tRNA校對中保有關鍵作用;The C-Ala domain of human cytoplasmic AlaRS preserves an essential role in tRNA editing

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Title:	人類細胞質AlaRS的C-Ala在tRNA校對中保有關鍵作用;The C-Ala domain of human cytoplasmic AlaRS preserves an essential role in tRNA editing
Authors:	劉世暘;Liu, Shih-Yang
Contributors:	生命科學系
Keywords:	丙胺酸-tRNA合成酶;tRNA;C-Ala;tRNA結合區域;DNA結合區域;胺醯化;校對;Alanyl-tRNA synthetase;tRNA;C-Ala;tRNA-binding domain;DNA-binding domain;aminoacylation;editing
Date:	2025-08-04
Issue Date:	2025-10-17 11:25:50 (UTC+8)
Publisher:	國立中央大學
Abstract:	Aminoacyl-tRNA synthetases (aaRSs) 是一群普遍存在的必要酵素，負責將特定胺基酸接到其對應的tRNA 上。合成出的胺基酸-tRNA (aminoacyl-tRNA) 會被用於蛋白質合成，是蛋白質轉譯的關鍵步驟。除了參與蛋白質合成之外，多數真核生物 aaRSs 在演化上透過獲得額外的 N 端或 C 端區域，來拓展新的非轉譯功能，這些區域通常並不參與aaRSs的典型功能。有趣的是，在 20 種 aaRSs 中，只有Alanyl-tRNA synthetase (AlaRS) 在古菌、細菌以及真核生物中，都保留了同樣的區域結構，並未獲得新的功能區域。然而，這並不意味著AlaRS並未獲得新的功能，人類AlaRS (HsAlaRS) 的C端區域 (C-Ala) 在作為單獨存在的剪接變體時，能夠與DNA結合並調控基因表現。在大腸桿菌中，C-Ala 協助 tRNAAla 結合、胺醯化tRNAAla、校對tRNAAla上的胺基酸，與協助AlaRS形成二聚體；而在人類中，C-Ala 已不再參與胺醯化，取而代之獲得了與 DNA 結合的能力。在本研究中，我們發現，儘管人類 AlaRS 的 C-Ala 有著功能上的差異，但其仍保有與 tRNA 結合的能力。值得注意的是，刪除 C-Ala 會顯著降低 AlaRS 的校對效率，幾乎完全喪失水解錯誤接上之 Gly-tRNAAla 的能力。此外，我們發現人類 C-Ala 在胺醯化過程中的重要性已變弱。為了補償 C-Ala 在胺醯化時原本協助 tRNA 結合的角色，人類 AlaRS 的 aminoacylation domain 獲得了新的 tRNA 結合能力。這些結果顯示，儘管 C-Ala 區域演化出新的功能，其仍參與 tRNAAla 的校對，暗示其在演化上保留並未因功能分化而成為獨立基因的原因，並揭示 AlaRS 與 tRNA 結合的高度可塑性。;Aminoacyl-tRNA synthetases (aaRSs) are a ubiquitous family of essential enzymes that catalyze the attachment of specific amino acids to their corresponding tRNAs, producing aminoacyl-tRNAs that are utilized by the ribosome during protein synthesis. Beyond their canonical roles in translation, many eukaryotic aaRSs have acquired additional N- or C-terminal domains that could expand their non-translational functions. Interestingly, alanyl-tRNA synthetase (AlaRS) is the only one that preserves a conserved architecture across all three domains of life. However, this evolutionary conservation does not preclude the acquisition of novel functions. The C-terminal domain of human AlaRS (Hs C-Ala) when expressed as a splicing variant in a freestanding form, exhibits DNA-binding activity and has been implicated in transcriptional regulation. In this study, we demonstrate that despite its functional divergence, the Hs C-Ala retains a strong binding affinity for its cognate tRNA. Notably, deletion of the C-Ala domain severely impairs the editing function of the enzyme, leading to a near-complete loss of hydrolytic activity against misacylated Gly-tRNAAla. Interestingly, to compensate for the diminished tRNA-binding role of C-Ala in aminoacylation, the aminoacylation domain of human AlaRS has evolved a stronger intrinsic affinity for tRNA. These findings suggest that while human C-Ala has acquired novel functions, it remains critical for maintaining the enzyme′s editing activity. This underscores an evolutionary constraint to preserve C-Ala within AlaRS despite its functional repurposing. Overall, our study highlights the evolutionary plasticity of AlaRS, particularly in adapting tRNA-binding strategies while integrating new regulatory roles.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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