Aminoacyl-tRNA synthetase (aaRSs) 是一群參與蛋白質合成的必要酵素，負責辨識並將特定的胺基酸接到其相應的tRNA上，是維持蛋白質轉譯準確性的關鍵步驟。本篇論文探討Histidyl-tRNA synthetases (HisRS) 與Prolyl-tRNA synthetases (ProRS) 在不同生物中如何與tRNA進行共演化，改變其結構以及辨識tRNA的機制以對應非典型tRNA。在一種嗜熱古菌Nanoarchaeum equitans中，由於缺乏RNase P修飾tRNA的5′端，且基因轉錄出的pre-tRNAHis並未帶有前導序列(Leader sequence)，此種古菌的tRNAHis有別於其他物種，帶有5′三磷酸(ppp-tRNA)而非單磷酸。我們發現其HisRS (NeHisRS) 與典型HisRS類似，仍然偏好帶有5′單磷酸 (p-tRNA)的tRNAHis，且重度依靠G-1位點來辨識tRNAHis。特別的是，與典型原核生物HisRS不同，NeHisRS能夠有效地胺醯化帶有A73或C73的tRNAHis，顯示其對辨別tRNAHis的專一性有所放寬。ProRS存在兩種結構形式，分別是原核生物型(P-type)，和真核生物/古菌型(E-type)，通常細菌只會帶有一個P-type ProRS。有趣的是，蘇雲金芽孢桿菌 (Bacillus thuringiensis) 同時帶有這二種ProRS，分別為BtProRS1 (P-type) 與BtProRS2 (E-type)。我們發現，BtProRS1與BtProRS2雖同能識別P-type tRNAPro，但採用不同的識別機制，且BtProRS2對halofuginone (HF)及環境壓力的耐受性更強，顯示帶有第二種ProRS的演化優勢。在弓形蟲 (Toxoplasma gondii)中，單一一種E-type ProRS (TgProRS) 能胺醯化細胞質(E-type)及頂質體(P-type)兩種具有不同識別元素的tRNAPro。TgProRS的motif 2 loop突變會選擇性影響不同tRNA的識別。此外，細胞質tRNAPro對HF的敏感度高於頂質體tRNAPro。這些發現揭示aaRS如何透過不同識別策略因應tRNA多樣性，深化對酵素與tRNA共同演化的理解，並為設計具選擇性的抗微生物療法提供理論依據。;Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ensure translational fidelity by specifically recognizing and aminoacylating their cognate tRNAs. This dissertation explores the adaptive recognition mechanisms of histidyl- and prolyl-tRNA synthetases (HisRS and ProRS) in diverse organisms, highlighting structural and functional evolution in response to noncanonical tRNA features. In Nanoarchaeum equitans, which lacks RNase P and transcribes leaderless tRNAs with 5′-triphosphates (ppp-tRNAs), we show that its HisRS (NeHisRS) maintains a strong preference for tRNAHis with a 5′-monophosphate (p-tRNAHis) and relies heavily on G-1 for substrate recognition. Unlike typical prokaryotic HisRSs, NeHisRS charges tRNAHis with A73 or C73 with similar efficiency, indicating relaxed specificity for the discriminator base. ProRSs exist in two structural forms: prokaryotic (P-type) and eukaryotic/archaeal (E-type). In Bacillus thuringiensis, both types coexist. Although BtProRS1 and BtProRS2 recognize the same P-type tRNAPro, they use distinct mechanisms. BtProRS2 also shows greater resistance to hf and stress conditions, suggesting an adaptive advantage. In Toxoplasma gondii, a single E-type ProRS (TgProRS) aminoacylates both cytosolic and apicoplast tRNAPro isoacceptors with distinct identity elements. Mutations in TgProRS’s motif 2 loop selectively impact substrate recognition. Additionally, cytosolic tRNAPro charging is more sensitive to halofuginone than its apicoplast counterpart. These findings reveal how aaRSs evolve distinct recognition strategies to accommodate tRNA diversity, offering insights into enzyme–substrate coevolution and informing strategies for targeted antimicrobial development.
