從結構和能量觀點探討耐金屬貪銅菌之金屬調節轉錄因子 CupR 與 DNA 結合的特異性

Abstract

CupR 是來自耐金屬貪銅菌 (Cupriavidus metallidurans CH34) 之金屬調節轉錄因子 (metalloregulatory transcription factor)，能夠調控其操縱子之基因表現與否。CupR 二聚體受到金屬離子所引發的構型變化，將導致操縱子轉錄調控區塊 (operator/promoter site) RNA 聚合酶結合位 -35 和 -10 區塊旋轉至同一平面上，因而活化轉錄作用。然而至目前為止，CupR 與其專一性啟動子區域結合的細節尚未被釐清。 為了瞭解 CupR 對其特異性 (specific) DNA 序列之辨識以及二者結合對蛋白質二級結構和 DNA 構型的變化，本研究利用電泳遷移率實驗 (Electrophoretic Mobility Shift Assay, EMSA)、恆溫滴定微量熱實驗 (Isothermal Titration Calorimetry, ITC)、圓二色光譜分析實驗 (Ciruclar Dichroism, CD) 對 CupR 與不同序列 DNA 之結合特性做比較探討，再藉由結構模擬以及分子對接技術 (Molecular Docking) 分析 CupR 對相似序列之辨識差異。CupR 與 DNA 之結合能力藉由電泳遷移率實驗分析，顯示 CupR 與其轉錄調控區塊 (PcupA) 的結合能力相較於非特異性核酸序列明顯較強，且亞金離子存在之環境下使二者結合能力提升。CupR 與 DNA 結合過程之熱力學參數，指出 CupR 與 PcupA 的結合過程中所量測得到之熱焓與亂度皆為正值，說明 CupR 主要是以氫鍵穩定與 PcupA 結合。經由 CupR 二聚體模擬結構與其轉錄調控區塊 PcupA 所建構的複合體 (complex) 與文獻所提供之同源蛋白質 CueR-DNA 結構作比較，結果顯示 CupR-DNA 其中三個參與交互作用的鹼基可能對於 CupR 辨識並結合專一性序列重要。

CupR, the metalloregulatory transcription factor from Cupriavidus metallidurans strain 34, regulates gene expression of metal-resistance operon. When the dimeric CupR coordinates to a metal ion, the metal-CupR complex causes an allosteric underwinding of the DNA at the operator/promoter site, which realigns the -35 and -10 sequences of the promoter. Subsequently, RNA polymerase can contact the promoter sequences leading to transcription activation. So far the detail recognition of CupR with its specific DNA-binding sequences remains unknown. In this research, we study the binding properties of CupR toward specific or non-specific DNA sequences through EMSA (Electrophoretic Mobility Shift Assay), ITC (Isothermal Titration Calorimetry) and CD (Circular Dichroism Spectroscopy). Furthermore, homology modeling and docking methods were used to investigate the recognition modes between CupR and various DNA sequences. Data from EMSA reveals that CupR posesses best binding ability with promoter sequences (PcupA) compared to others. Moreover, chelation of CupR with Au+ ion makes CupR binding to PcupA stronger. Additionally, the ITC thermodynamic parameters of CupR bound to promoter sequences (PcupA) shows a very negative enthalpy and a negative entropy effect which indicating that formation of CupR and PcupA is mainly via hydrogen-bonding. Molecular models of CupR in complexes with various DNAs are analyzed by NucPlot and reveal that three bases may be important for the recognition between CupR and promoter sequence.