以結構生物學探討雙重功能轉錄調節因子MerR 調控轉錄作用之機制

Structural analysis of transcriptional regulator MerR in complex with operator DNA and RNA polymerase holoenzyme

林佳陞(Chia-Sheng Lin)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102813

摘要

細菌在漫長的演化過程中產生許多可以對抗環境中有害物質的生理機轉其中不乏包含對於機體有相對高毒害性的汞金屬。這些對抗有毒物質的相關蛋白質或者是機轉通常都位在特定的操縱子(operon)中，像是汞抗性操縱組(mer operon)。汞抗性操縱組主要的功能是在幫助細菌對抗環境中汞金屬相關的有毒物質像是有機汞或者是二價汞離子。當汞抗性轉錄調節因子(MerR)偵測到環境中有二價汞離子或者是有機汞存在的時候， MerR會促使汞抗性操縱組轉錄汞抗性相關的蛋白，而這一切轉錄的起始都是受到MerR的嚴密調控。MerR與一般轉錄調節因子(transcriptional regulator)最大的差異在於其同時具有抑制與促進轉錄的功能。造成MerR擁有雙重調控轉錄的功能的主因是因為汞抗性操縱組的轉錄調控區塊(operator/promoter)的-10區域(-10 element)和-35區域(-35 element)之間較一般轉錄調控區塊多了兩個鹼基對，進而導致-10區域和-35區域不會座落在相同的平面上，使得σ因子(sigma factor)無法同時辨識-此二區域，造成轉錄無法進行。由過往的生化學實驗得知，當環境中有機汞或者是二價汞離子不存在時， MerR會結合到鄰近-35區域的結合位並扮演抑制物(repressor)的角色。當有機汞或者是無機汞與MerR結合時， MerR會發生構型改變使得-10區域和-35區域會被扭轉至同一個平面上，使得σ因子得以進行辨識並啟動轉錄。過往的研究就曾將MerR與二價汞離子進行共結晶並成功解析結構，透過晶體結構我們得知而價汞離子會結合至MerR序列中高度保留、由三個半胱氨酸(cysteine)組成的中心並形成三配位的平面三角結構。然而對於有機汞是如何與MerR結合並造成構型變化的詳細機轉仍然未被解開，而過去的研究中也僅指出位於pDU1358 MerR C端的17個氨基酸對於結合有機汞十分重要，但是並無任何關於交互作用的結構資訊。除此之外，目前對於RNA聚合酶全酶(RNA polymerase holoenzyme)如何和有機汞結合態的MerR以及汞抗性操縱組的轉錄調控區塊進行交互作用的詳細機轉也仍屬未知。因此，我的研究目標是希望透過結構解析，嘗試了解MerR調控汞抗性操縱組轉錄活性的結構基礎。
本研究預期透過X-光晶體繞射(X-ray crystallography)以及冷凍電子顯微鏡(Cryogenic Electron Microscopy)二種技術進行解構。首先，我們先表現並純化pDU1358 MerR。接著我們依序加入含有汞抗性操縱組轉錄調控區塊的去氧核醣核酸、有機汞、RNA聚合酶全酶，並對此複合體進行結構解析。鑒於冷凍電子顯微鏡得到的電子密度圖並未顯示pDU1358 MerR的存在，因此我們設計一個報導基因檢測法(reporter assay)來檢測pDU1358 MerR是否會和有機汞進行交互作用。報導基因檢測法結果顯示pDU1358 MerR確實會和與機汞發生交互作用，但是親和力低於Hg(II)。我們也將汞抗性操縱組的轉錄調控區塊中連結區段的長度由19延長至20個個核苷酸、並將汞抗性操縱蛋白結合位(MerR binding site)往-35區域以及-10區域分別移動一個核苷酸並透過報導基因檢測此改變對轉錄活性的影響。結果顯示，改變汞抗性操縱組轉錄調控區的架構會對MerR活化轉錄的效率產生嚴重的影響。

關鍵字

汞抗性操作組 ； 轉錄調節因子 ； MerR ； RNA 聚合酶 ； 冷凍電子顯微鏡 ； 報導基因檢測法

並列摘要

Bacteria possess a wide variety of resistance mechanisms to counteract different environmental toxins such as the highly reactive mercuric species. The components of these mechanisms are often encoded by specific operons. The mercury resistance (mer) operon confers bacterial resistance to inorganic and organomercurial compounds. Upon sensing the presence of mercuric ion (Hg(II)) or organomercury (HgR) in the environment, the mer operon will be activated to express resistance proteins. The transcription of mer operon is under tight control by the dual function transcriptional regulator MerR. The key difference between canonical and the promoter of mer operon is that the distance between -10 and -35 region is 2-base longer in the latter, which causes -10 and -35 region lying on opposite sides of the operator DNA, making them difficult for interacting with the sigma factor of the RNA polymerase holoenzyme simultaneously and thereby suppressing transcription activation. Biochemical studies revealed that, in the absence of an activator, the apo MerR acts as a repressor by binding to the recognition site close to the -35 region. On the contrary, MerR undergoes a conformational change upon Hg(II) or HgR binding, which induces DNA twisting to bring the -10 and -35 region to the same side of DNA to allow efficient sigma factor recognition and transcription activation. The crystal structures of Hg(II)-bound MerR reveal that Hg(II) is coordinated by three highly conserved cysteine residues arranged in a trigonal planar geometry. Despite the rather detailed understanding of how MerR interacts with Hg(II), the interaction between MerR and HgR has not been structurally elucidated. Previous biochemical studies indicate that the C-terminal 17 amino acid residues of pDU1358 MerR, an unique MerR family member capable of interacting with HgR, might play an important role in HgR binding. However, the mechanistic detail has remained mysterious. Besides, the structures of the complexes formed by MerR, operator DNA, and RNA polymerase holoenzyme have yet to be determined. Thus, the objective of my thesis research is to understand structurally how HgR-bound MerR modulates the association between mer operator-promoter DNA and RNA polymerase holoenzyme to activate transcription.
To this end, we seek to characterize pDU1358 MerR in complex with operator DNA and HgR by X-ray crystallography and cryogenic electron microscopy (cryo-EM). We first overexpressed and purified pDU1358 MerR, followed by the addition of HgR and operator DNA for co-crystallization and X-ray diffraction analysis. In addition, cryo-EM was used as an alternative approach for structure determination. The electron density map obtained by cryo-EM showed that pDu1358 MerR was absent in the complex. Thus, we designed a reporter assay to examine whether pDu1358 MerR may interact with phenyl mercury acetate via the three highly conserved cysteine residues. The reporter assay demonstrated that, albeit less effective than Hg(II), transcription activation can indeed be observed upon the addition of phenyl mercury acetate, indicating that pDu1358 MerR interacts with both Hg(II) and HgR. Also, we examined the effects of varying the spacing between -10 and -35 promoter elements and MerR binding site mutants on transcription efficiency. The results indicated that alteration of promoter architecture and mutating metal coordinating residues of MerR would severely affect the transactivation activity of pDu1358 MerR.

並列關鍵字

mer operon ； transcriptional regulator ； MerR ； RNA polymerase ； cryo-EM ； reporter assay

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