秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla;Caenorhabditis elegans mitochondrial AlaRS recognizes a T-armless tRNAAla through a non-traditional mode

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題名:	秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla;Caenorhabditis elegans mitochondrial AlaRS recognizes a T-armless tRNAAla through a non-traditional mode
作者:	瑪希塔;Imamsari, Masita
貢獻者:	生命科學系
關鍵詞:	AlaRS;tRNA胺醯化;C-Ala結構區;辨識元素(identity element);tRNAAla;AlaRS;aminoacylation;C-Ala domain;identity element;tRNAAla
日期:	2020-12-16
上傳時間:	2021-03-18 16:50:25 (UTC+8)
出版者:	國立中央大學
摘要:	Aminoacyl-tRNA synthetases（aaRSs）是一群普遍存在的酵素，它們的主要功能是將特定胺基酸接到相對應的tRNA，例如alanyl-tRNA synthetase（AlaRS）會將Ala接到tRNAAla 。 AlaRS具有四個重要的結構區，來維持完整的功能，包括催化區、tRNA結合區、編輯區、C-Ala區。這四個結構區是高度保留的，並且提供了完整的tRNA胺醯化及編輯能力。有趣的是，秀麗隱桿線蟲的粒線體AlaRS（CeAlaRSm）缺少C-Ala區，且其對應的tRNAAla缺乏TΨC，即便如此，CeAlaRSm仍然能精確地辨認tRNAAla上的辨識元素G3:U70配對。然而，我們的實驗結果發現CeAlaRSm只能有效胺醯化微型tRNAAla (microhelix-Ala)，卻無法有效胺醯化完整的tRNAAla，如果將線蟲細胞質AlaRS（CeAlaRSc）的C-Ala區與CeAlaRSm融合產生融合?，這個融合?在in vivo和in vitro的情形下皆能更有效地胺醯化tRNAAla。然而，從CeAlaRSc中移除C-Ala區後，其胺醯化功能將產生缺陷。另外，一般AlaRS的tRNA結合區都含有兩個高度保留的胺基酸Asn和Asp，這二個胺基酸主要用來辨識tRNAAla 中的辨識元素(identity element) G3：U70。然而，CeAlaRSm在相對應位置的胺基酸卻是Gly和Glu。若將Gly322或Glu420突變為Ala，將導致酵素的胺醯化活性降低(in vitro)，以及失去提供酵母菌AlaRS剔除株存活所需的胺醯化能力(in vivo)，這結果表示，雖然CeAlaRSm沒有高度保留的胺基酸Asn及Asp，Gly及Glu仍然參與tRNAAla的G3:U70辨認。我們的結果顯示，CeAlaRSm是一種非典型的AlaRS，可通過非傳統的模式來辨認缺乏TΨC的 tRNAAla。;Aminoacyl-tRNA synthetases (aaRSs) are a family of ubiquitously expressed enzymes, attaching a specific amino acid to its corresponding tRNA, such as alanyl-tRNA synthetase (AlaRS) attaching alanine to tRNAAla. AlaRS consists of four important domains to work properly, including catalytic domain, tRNA-binding domain, editing domain, and C-Ala domain. Those four domains are highly conserved and support the full function of tRNA alanylation. Interestingly, C. elegans mitochondrial AlaRS (CeAlaRSm) lacks C-Ala domain. Our results showed that CeAlaRSm retains its tRNA specificity, but poorly recognizes a full-length tRNAAla in vivo and in vitro. In addition, we showed that fusion of the C-Ala domain of C. elegans cytoplasmic AlaRS (CeAlaRSc) to CeAlaRSm results in a fusion enzyme that can more efficienty charge tRNAAla in vivo and in vitro. On the other hand, deletion of the C-Ala domain from CeAlaRSc yielded a truncated enzyme defective in aminoacylation. Unlike canonical AlaRSs, which contain two highly conserved amino acid residues, Asn and Asp for recognition of the canonical identity element G3:U70 in the tRNAAla. CeAlaRSm contains Gly and Glu which at the corresponding positions. Mutation of G322 to A or E420 to A led to an enzyme defective in aminoacylation in vitro and complementation in vivo. Our results suggest that CeAlaRSm is a non-canonical AlaRS that recognizes a T-armless tRNAAla through a non-traditional mode.
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