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標題: | T4噬菌體DNA擬態蛋白質Arn抑制細菌類組蛋白質H-NS之DNA結合活性研究 The T4 Phage DNA Mimic Protein Arn Inhibits the DNA-binding Activity of the Bacterial Histone-like Protein H-NS |
作者: | Chun-Han Ho 何俊翰 |
指導教授: | 王惠鈞(Andrew H.-J. Wang) |
關鍵字: | 分子擬態,DNA擬態蛋白質,抗細菌(宿主)防禦機制,抗基因沉默,類核體, molecular mimicry,DNA mimic protein,anti-bacterial defense system,anti-gene silencing,nucleoid, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | DNA是一般生物的遺傳物質,經由一些DNA相關之功能性蛋白質,如轉錄因子或是DNA結合蛋白質可以調控DNA序列中所蘊涵的遺傳訊息之表達。大約在十年前,有一類的蛋白質稱之DNA擬態蛋白質被發現,他們在蛋白質結構上所展現出的特殊形態是模擬DNA的總體形狀以及DNA表面負電荷排列狀態,使其他與DNA作用的蛋白質無法區別而與之作用,進而達到抑制某些特定DNA相關功能性蛋白質的活性與功能,因此DNA擬態蛋白質往往參與著很多重要的細胞生理調控以及存在於許多尚未被發現的調控機制當中。
為了找尋T4噬菌體中DNA擬態蛋白質,在本篇研究中,先鎖定具有DNA擬態蛋白質的通性:分子量較小與等電點較低的蛋白質做為我們初篩選的標的蛋白質進行分析。經篩選後發現,其中Arn這個蛋白質很有可能是一個尚未被發現的DNA擬態蛋白質;於是利用結構生物學的方法,解析Arn的3D立體結構。非常有趣並令人振奮地,從Arn的3D立體結構可以發現:Arn二聚體表面帶負電的胺基酸分佈與雙股DNA帶負電的磷酸基團分佈極為類似,此結果強烈暗示著Arn可能是一個DNA擬態蛋白質。除此之外,Arn二聚體的大小與形狀也與另一個已知的DNA擬態蛋白質Ocr之二聚體相類似。為了解T4噬菌體Arn在大腸桿菌體中與哪個蛋白質有交互作用,於是進行了蛋白質沉澱技術,並發現細菌類組蛋白質H-NS可與T4噬菌體蛋白質Arn進行蛋白質-蛋白質專一性結合。 而這個初步的發現暗示著可能隱藏著全新的調控機制。由於T4噬菌體感染其染宿主大腸桿菌時會遭受到宿主的防禦系統攻擊,如:細菌體內會表現一些小型的DNA結合蛋白質結合外來或噬菌體DNA並造成抑制其基因表現,其中細菌類組蛋白質H-NS就是此類蛋白質的成員之一。因此T4噬菌體為了能成功地感其染宿主並在菌體內快速完成感染週期,T4噬菌體勢必要演化出抵抗宿主此類的防禦系統。而T4噬菌體利用DNA擬態蛋白質來抑制細菌類組蛋白質H-NS與噬菌體DNA結合,對噬菌體來說,則很可能是一個很有效率抵抗宿主的機制。 因此為了更進一步證實與了解T4噬菌體蛋白質Arn的功能是否與我們的假設相符,利用競爭性的電泳遷移率改變分析法,可以發現Arn可以競爭並取代DNA片段與DNA結合蛋白質H-NS結合;在電腦3D結構的模擬中更顯示著Arn二聚體利用模擬DNA電性分佈的一側和H-NS原本與DNA結合區域進行結合,進而阻擋了DNA與H-NS的結合。此外;在胞外基因表現分析與電子顯微鏡影像分析中更證實Arn可以阻擋H-NS對基因抑制的活性。 在此研究中,我們在T4噬菌體中發現了一個全新的噬菌體-宿主的對抗機制:一個全新的DNA擬態蛋白質Arn,它藉由模擬DNA表面負電荷分佈,與細菌類組蛋白質H-NS結合並阻擋了其DNA結合位,而達到阻擋H-NS對基因抑制的活性。 Organisms using DNA as their hereditary substance, the genetic information of these DNA sequence can be expressed and regulated by DNA-related functional proteins, such as transcriptional factors and DNA-binding proteins. Around a decade ago, a new category of control factors of DNA-related functional proteins called the DNA mimic protein had been identified. The DNA mimic protein can inhibit and/or regulate the function and activity of DNA-related functional proteins by its DNA-like shape and unique surface negative charge pattern. Thus, DNA mimic proteins are involved in certain important cellular processes and may be involved in many undiscovered regulation mechanisms. To identify the DNA mimic protein in T4 phage, we focused on the proteins which have the properties found in known DNA mimic proteins: small protein size and low isoelectric point (pI). After the sequence analysis, we found that anti restriction nuclease (Arn) protein may be a DNA mimic protein. By using structural approaches, the crystal structure of Arn was determined. Interestingly, the negative charge distribution of Arn dimer surface is similar to the phosphate group distribution on DNA, implying Arn dimer could act as a DNA mimic. Furthermore, the size and shape of Arn dimer is similar to the DNA mimic protein overcome classical restriction (Ocr). To identify the interaction partner of T4 phage Arn in Escherichia coli (E. coli), the His-pull-down was used and we further discovered that bacterial histone-like nucleoid structuring (H-NS) protein can interact with Arn specifically. Arn/H-NS interaction reveals a novel regulation mechanism. When infecting the E. coli, T4 phage encounters attacks from host’s defense systems. For example, bacteria express some small DNA-binding proteins to bind and entangle foreign or phage DNAs, inducing the gene silencing effect. H-NS is a member of these kind of protein. Thus, in order to infect and replicate in E. coli successfully, T4 phage has to evolve some strategies to overcome the gene silencing effect from H-NS. Therefore, using a DNA mimic protein to inhibit H-NS is a straightforward way for T4 phage. To confirm our hypothesis in this undiscovered function of Arn, electrophoresis mobility shift assay (EMSA) was used and demonstrated that Arn competes with bacteria as well as phage DNA fragments for binding to H-NS. Computer modeling analysis revealed that Arn dimer competes with DNA to interact with the H-NS DNA binding domain via its negatively-charged side. Additionally, in vitro gene expression and electron microscopy analyses further indicated that Arn antagonizes the gene-silencing effect of H-NS on the reporter gene. In summary, we discovered a novel mechanism for phage-bacteria battle from T4 phage, which employs the DNA mimic protein Arn to counteract the H-NS gene-silencing effect by its DNA-like surface. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54388 |
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