在原子層級解析 RNA E-loop Motif的摺疊與演化

Abstract

RNA是令人驚奇的生物分子，在生物裡扮演的角色與其立體構造息息相關，愈深究RNA的立體構造，愈能揭開RNA在生物內的催化功能及機轉，他的骨架具有彈性而易彎折，因此RNA的立體結構具有局部多變異的特性。重複出現的RNA構造稱為RNA模組，已知的RNA模組有Tetraloop、Kink-turn、E-loop等，這些構造被認為與分子立體結構的穩定及生化反應催化活性息息相關。我的研究主題為RNA E-loop模組，它是由雙股RNA組成，其中一股的結構與A-form相似，另一股的骨架則扭曲成S型。在過去文獻中，E-loop模組被發現於23S rRNA的內層、RNA三股交叉點及第II型內含子中，並指出此結構可以和延伸因子－G（EF-G）結合。我利用細菌及古生菌的核糖體三維結構，進行迴圈式地生物資訊學構造探勘，分析並分類我所發現的E-loop motif。依據我所定義的參數含括對構型的新定義，我將E-loop模組分類為成熟型、過渡型及原始型。我利用統計E-loop模組的演化共變性，觀察它在三維空間裡局部構型的多變性，並進行分子動力的引導計算模擬。我的研究成果總結如下：(1) 我利用生物資訊學分析方法進行23S rRNAs的構造探勘，有超過60% 的E-loop模組是全新發現，在過去文獻中未被報導。(2) 我所發現的E-loop模組具有構型的多樣性。(3) 我利用結晶構造解析及分子動力的引導計算模擬，推演出E-loop模組在原子層級解析度下的折疊機制。(4) 我建立了E-loop模組的演化模型，由原始型的結構初始化，歷經過渡型，最後演化為成熟型的E-loop模組。 (5) 透過核醣體的構造探勘，我推論了E-loop模組是在選擇性壓力的驅使下，與核醣體共同演化。所以，RNA模組的熟成也是穩定核糖體整體結構和扮演生化反應的重要基礎元素。

RNA is amazing. Its pliable backbone deviates the RNA local structure plays important roles in many biological functions and enzymatic catalysis. The repetitive RNA structures, called RNA motifs such as Tetraloop, Kink-turn, E-loop, etc., are considered to be essential intrinsic elements that stabilize the RNA conformations and confer the RNA catalytic functions. Among many unique RNA motifs, the RNA E-loop motif is in a helix form with one stand in an A-form conformation, and the other in an S-shape. The RNA E-loop motif is observed in the core of the 23S rRNA, in the three-way junctions of the 23S rRNA, in the catalytic RNA of group II introns, and is critical for anchoring the elongation factor G (EF-G) of the ribosome. Here we iteratively structural mine the E-loop motif within the bacterial and archaeal ribosomes. We focus on analyzing and classifying the three dimensional structures of the RNA E-loop motif. We statistically compute the phylogenic covariations, conformationally inspect the RNA local structural deviations, and systematically analyze the intra-molecular interactions of the E-loop motifs. We also perform the directed MD (molecular dynamics) simulations on the folding of the E-loop motif. Here we show that (i) more than 60% of the E-loops are uncovered, (ii) RNA E-loop motif accommodates the local structural deviations, (iii) we postulate the reaction coordinates of the E-loop motif folding at atomic resolution, (iv) the evolution of the RNA E-loop motif involves with three periods, which we define, are mature, intermediate, and primitive period, and (v) in each period of the E-loop motif is coped with the evolution of the large subunit of the ribosomes.