以結構學探討汞離子誘發雙重功能轉錄調節因子MerR構型變換如何調控轉錄作用

Abstract

細菌演化出對抗不同毒性金屬的操作組(operon)來在嚴苛的環境下生存，而其中的汞抗性操作組(mer operon)是最早被發現研究重金屬防禦系統之一。 汞抗性操作組可以製造對抗二價汞或是有機汞所需的去毒性蛋白和通道蛋白。汞抗性操作組受到雙重功能的轉錄調節因子(transcriptional regulator)MerR嚴密調控，在環境中沒有汞的存在下，原態（apo）的MerR會結合上轉錄調控區塊（operator/promoter），做為抑制子(repressor)的角色，反之當感應到環境中有汞存在時，MerR轉換為活化子（activator）的角色，透過本身的金屬結合區塊(metal binding domain)結合上汞金屬後，所產生構型轉變來帶動並扭曲（distort）汞抗性操作組DNA，使轉錄作用可以開始進行。然而，並沒有結構的資訊關於MerR是如何讓RNA聚合(RNA polymerase)順利結合上啟動子(promotor)序列並開始轉錄汞抗性操作組。所以本研究的目標是藉由得到MerR轉錄因子和RNA聚合一同結合上汞抗性操作組，所形成三者複合體的結構資訊來了解這個MerR家族轉錄因子是如何調控RNA 聚合轉錄這段被MerR扭曲的汞抗性操作組DNA。 到目前我們已經成功使用大腸桿菌系統表現並純化出全長的RNA聚合全酶(holoenzyme)，RNA 聚合全酶為分子量極大的蛋白，由各一個β和β′次單位、兩個α次單位和一個ω次單位組成核心聚合酶（core enzyme），核心聚合酶最後會結合上σ因子形成具有功能且分子量約450 kDA的RNA 聚合酶全酶。我們在RNA 聚合酶α次單位上帶有10X His tag 和蛋白酶切位，在固定化金屬離子親和層析法純化後，使用蛋白酶切除His tag後，通過後續兩次離子交換層析增進蛋白純度後以膠體過濾層析管柱確認後，再和使用固定化金屬離子親和層析法和離子交換層析純化後的σ因子進行組裝，最終使用膠體過濾層析管柱得到高純度和正確構型的RNA 聚合酶全酶。我們的MerR蛋白會使用肝素(heparin)親和性管柱和膠體過濾層析管柱純化後，依序加入我們設計好的汞抗性操作組DNA和純化完成的RNA 聚合酶。等形成穩定複合體後，再次使用膠體過濾層析法，確認三者能形成一個約500 kDa穩定複合體後，利用戊二醛(glutaraldehyde)將我們的蛋白複合體進行交叉鍵接(crosslinking)反應，此步驟可以讓蛋白樣品在使用液態氮冷凍後維持在正確的複合體構型，不會因為冰晶形成破壞各次單位原先的交互作用。我們也可以設計不同的抗性操作組DNA或是將MerR轉錄因子處於活化子或是抑制子的不同條件組合下，利用冷凍電子顯微鏡技術，獲取RNA 聚合酶位於不同的構型，來幫助我們了解MerR轉錄因子是如何幫助轉錄作用進行的。

All bacteria possess resistance systems (operons) in order to survive in environments containing toxic metal ions. The bacterial mercury resistance operon (mer operon) is one of the earliest discovered and best characterized toxic metal defense systems. The mer operon encodes proteins that can detoxify and transport inorganic mercury (Hg2+) and organic mercury compounds. Expression of the mer operon proteins is under control by the dual-function transcriptional regulator MerR. In the absence of Hg2+, apo MerR binds to the mer operator/promoter (o/p) region and acts as a transcriptional repressor. In contrast, Hg2+-binding triggers structural transition of the MerR dimer to induce conformational change of o/p DNA, which initiates transcription. Currently, no structural information regarding the interplays between MerR, RNA polymerase (RNAP) holoenzyme and mer o/p are available. The goal of my thesis research is to characterize the 3D structure of RNAP in complex with MerR and mer o/p. To this end, we have attempted to purify recombinant E.coli RNAP. The RNAP holoenzyme is about 450 kDa in size, containing a core enzyme formed by two α subunits, a single β, β′ and ω subunit, and one of the seven σ factors. We have obtained a RNAP construct with a protease recognition site introduced between at the N-terminal 10XHis tag and α subunit. After initial purification by immobilized metal ion affinity chromatography (IMAC), protease may be used to remove the N-terminal 10XHis tag. σ factor can be purified in order of IMAC, ion exchange chromatography, and size exclusion chromatography. The full length RNAP holoenzyme can be reconstituted by mixing the core enzyme with σ factor followed by size exclusion chromatography. MerR protein is purified in order of affinity chromatography, and size exclusion chromatography. For cryo-EM structural analysis, purified MerR protein (with or without mercury) was first incubated with mer o/p before the addition of RNAP holoenzyme. The ternary supramolecular complex was confirmed by size exclusion chromatography. Then, RNA polymerase holoenzyme ternary complex was crosslinked by glutaraldehyde to prevent liquid nitrogen treatment-induced protein ternary complex dissociation. After crosslinking, protein sample concentration was adjusted to 1 mg/ml for cryo-EM data collection. We can use different mer o/p DNA sequence design combined with Hg2+ to capture RNA polymerase in different stages during transcription initiation.