古生菌硫磺礦硫化葉菌蛋白質Sso7c4與去氧核糖核酸作用機制之研究

古生菌硫磺礦硫化葉菌中的蛋白質Sso7c4 在溶液中以雙聚體的形式存在，一般相信其為染色體蛋白質，功能是參與去氧核醣核酸組裝或基因轉錄調控。我們利用X 光單晶繞射技術解析出Sso7c4 蛋白質的晶體結構，解析度為1.63 埃。而在這個蛋白質晶體的繞射數據中，蛋白質的C 端完全沒有電子密度，顯示C 端是相當動態的。利用螢光極化光譜量測Sso7c4 突變蛋白質(R11A,R22A,R11A/R22A) 與DNA 的結合常數並與野生型的Sso7c4 比較。實驗結果顯示Sso7c4 突變蛋白質 (R11A,R22A,R11A/R22A)與DNA 分子的結合力皆小於野生型，而其中蛋白質 R11A/R22A 幾乎不與DNA 作用。這證明了位於Sso7c4 上方表面的R11 與R22 兩個帶正電的胺基酸，有參與DNA 的結合。由電子顯微影像的分析，證實野生型 Sso7c4 主要是以架橋與彎曲兩種方式與DNA 作用。相反地C 端最末六個胺基酸被刪除的Sso7c4 蛋白質，在高濃度下，會將環狀的DNA 分子撐開，且沒有任何讓DNA 更緊密結實的現象。而綜合螢光極化光譜、電子顯微影像以及螢光共振能量轉移技術的數據，我們證明了Sso7c4 蛋白質動態且帶正電的C 端，不僅參與 DNA 分子結合，也會使DNA 分子彎曲。在前人的研究中，依照胺基酸序列的比對，將Sso7c4 歸類於類轉錄抑制蛋白質。綜合以上所有數據資料，我們利用代謝產物活化蛋白與DNA 複合體中，彎曲的DNA 分子結構來建構Sso7c4 與DNA 複合體的模型。更進一步利用螢光共振能量轉移實驗量測出與Sso7c4 結合的DNA 分子兩端的距離與模型中的距離吻合，驗證了Sso7c4-DNA 模型的準確度。螢光共振能量轉移數據同時也驗證了電顯影像分析的結果，野生型Sso7c4 會使得DNA 分子長度縮短(表示DNA 分子被蛋白質彎曲)，但是C 端刪除的蛋白質無法使DNA 分子長度改變。綜合以上結果，我們證明Sso7c4 蛋白質建構或組裝染色體DNA 或是參與基因轉錄的調控，主要是藉由架橋與彎曲兩種方式來與DNA 分子作用。

關鍵字

去氧核醣核酸結合蛋白；大分子結晶學；電子顯微術；瑩光共振能量轉移；瑩光極化光譜；去氧核醣核酸組裝

並列摘要

The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22¢ and R11¢/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wildtype Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested.

並列關鍵字

DNA binding protein ； X-ray crystallography ； electron microscopy ； FRET ； fluorescence polarization ； DNA packing

參考文獻

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古生菌硫磺礦硫化葉菌蛋白質Sso7c4與去氧核糖核酸作用機制之研究

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