Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92203
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dc.contributorBan-Yang Changen_US
dc.contributor張邦彥zh_TW
dc.contributor.author鄭笠綺zh_TW
dc.contributor.authorLi-Chi Chengen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-15T05:30:03Z-
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dc.identifier.urihttp://hdl.handle.net/11455/92203-
dc.description.abstract枯草桿菌的σA蛋白屬於主要的σ蛋白(primary sigma),具有4個保守性區域(region),每個區域可細分為2到4個次區域(subregion)。本研究所探討的三個鹼性胺基酸均位在σA蛋白1.2次區域。本實驗室曾經探討1.1次區域對sigma功能發揮所扮演的腳色,發現刪除σA蛋白N-端第1到103個胺基酸,也就是涵蓋σA蛋白1.1次區域及1.2次區域的103胺基酸Arginine,會極顯著地降低σA蛋白參與轉錄的活性。胺基酸序列比對發現1.2次區域中的這三個鹼性胺基酸R103、K107及R111皆為保留性胺基酸。同源模擬晶體結構更發現此三個鹼性胺基酸位在1.2次區域所形成的helix-loop-helix的構造上,而且它們均朝同一個方向。另外,同時含有這三個鹼性胺基酸以alanine取代的突變型sigA菌株Bacillus subtilis DB2HY7,和野生型菌株Bacillus subtilis DB2相比,不論是在固態或液態的2xSG培養基中,均有提早細胞溶解(cell lysis)的現象。本研究旨在探討此三個鹼性胺基酸的alanine取代對σA蛋白與其所組成的RNA聚合酶在轉錄功能的影響,以及這三個鹼性胺基酸的alanine取代造成細胞提早溶解的原因。本研究藉in vitro transcription分析,來觀察野生型及突變型σA所組成之RNA聚合酶,對孢子形成初期會控制細胞自溶(autolysis)之spo0基因的σA-type 啟動子(promoter)轉錄活性的影響。希望藉此了解此三個鹼性胺基酸對σA功能發揮的重要性、以及造成此sigA突變株於孢子形成初期產生自溶現象的詳細原因。In vitro transcription的分析結果顯示,突變型σA-RNA聚合酶對spo0啟動子DNA的轉錄活性,相較之下,皆低於野生型σA-RNA聚合酶對spo0 啟動子DNA的轉錄活性。特別是突變型σA-RNA聚合酶對spo0A啟動子DNA的轉錄活性,明顯低於野生型σA-RNA聚合酶對spo0A 啟動子DNA的轉錄活性。因此,造成B. subtilis DB2HY7提早溶裂的原因,可能源於突變型σA-RNA聚合酶對spo0A啟動子之較低轉錄活性。進一步利用EMSA探討σA-RNA聚合酶對spo0A啟動子DNA之結合能力。發現突變型σA-RNA聚合酶和野生型σA-RNA聚合酶與spo0A啟動子DNA之結合能力,並未有明顯的差異,推測突變型σA蛋白可能影響到σA-RNA聚合酶和spo0A啟動子DNA所形成封閉式複合體之穩定度或此複合體進一步形成開放式複合體的能力。zh_TW
dc.description.abstractThe primary σ factor of Bacillus subtilis, σA, comprises four conserved regions, each containing two to four subregions. It was found that deletion of 103 amino acid residues from the N terminus of the σA would affect the efficiency of promoter DNA binding of the truncated σA-RNA polymerase, the stability of the closed binary complex and the efficiency of open complex formation. Sequence alignment of σ factor has revealed three conserved basic amino acid residues, R103, K107, and R111, in the N-terminus of subregion 1.2 of Bacillus subtilis σA factor. On the basis of a homology model structure of B. subtilis σA-RNA polymerase derived from the crystal structure of Thermus aquaticus σA-RNA polymerase, Arg-103 and Lys-107 are located on the same side of the first alpha-helix of a 'helix-loop-helix' motif in subregion 1.2 and their side chains are on the same orientation with the side chain of Arg-111, which is the second residue of the subsequent loop. Moreover, when the σA-RNA polymerase is complexed with a fork-junction promoter DNA, Arg-103 and Lys-107 are predicted to interact with Pro-242 and Gly-243 of the beta subunit through van der Waals interaction, and Arg-111 with Lys-147 of the beta' subunit through hydrogen bonding. The recent work has demonstrated that replacement of the three basic amino acids with alanine would result in a early cell lysis phenotype of the mutant sigA strain, B. subtilis DB2HY7. This study is aimed to answer how can replacement of the three basic amino acids of the σA with alanine lead to lysis of the cells during early sporulation and what is the functional defect of the mutant σA during transcription. To fulfill this goal, both the wild-type (Wt) and mutant σA factors were overexpressed, purified and used to reconstitute with the purified core RNA polymerase. The reconstituted Wt and mutant σA-RNA polymerase holoenzymes were then used to transcribe in vitro five selected spo0 promoters, including spo0A, spo0B, spo0E, spo0F and kinB, which all belong to the phosphorelay of early sporulation. The results showed that the mutant σA-RNA polymerase is weaker on transcribing all of the spo0 promoters comparing with the Wt σA-RNA polymerase, especially on spo0A, of which the protein product, Spo0A, is a master DNA-binding regulatory protein and regulates directly or indirectly the expression of over 500 genes during the early stages of development. Since the Wt and mutant σA-RNA polymerases have similar binding activities on the spo0A promoter DNA as analyzed by electrophoretic mobility shift assay (EMSA). I infer that the mutant σA-RNA polymerase may be defective in forming a stable closed complex or in forming a functional open complex with at least the spo0A promoter.en_US
dc.description.tableofcontents中文摘要 1 Abstract 2 材料與方法 12 一、實驗材料 12 二、菌株、質體與DNA引子來源 12 三、實驗方法 12 (一)σA蛋白之大量生產 12 (二)σA蛋白之純化 12 (三)枯草桿菌σA蛋白1.2次區域胺基酸取代之突變型sigA基因的構築 13 (四)突變型σA蛋白之大量生產 14 (五)突變型σA蛋白之純化 14 (六)純化具有histidine標示之B. subtilis RNA聚合酶核心酵素 15 (七)蛋白質之定量 17 (八)SDS-PAGE及銀染分析蛋白 17 (九)染色體DNA之抽取 18 (十)spo0基因之啟動子DNA之製備與純化 18 (十一)質體之抽取 19 (十二)重組RNA聚合酶的體外轉錄活性(in vitro transcription activity)分析 19 (十三) φ29 噬菌體G3b以及spo0 gene啟動子DNA之純化與標定 20 (十四)枯草桿菌野生型與突變型σA-RNAP與spo0基因啟動子DNA結合能力之Electrophoretic mobility shift assay(EMSA)分析 20 結果 21 一、枯草桿菌σA蛋白純化 21 二、純化具有histidine標示之B. subtilis RNA聚合酶核心酵素 22 三、重組RNA聚合酶的體外轉錄活性(in vitro transcription activity)分析 22 四、枯草桿菌野生型與突變型σA-RNAP與spo0基因啟動子DNA結合能力之Electrophoretic mobility shift assay(EMSA)分析 23 討論 25 附圖 27 圖一、B. subtilis孢子時期中自溶素(autolysins)切割肽聚醣層(peptidoglycan)之切位圖 27 圖二、解釋枯草桿菌DB2HY7早期細胞裂解和在枯草桿菌phosphorelay信號轉導途徑之模型 28 圖三、枯草桿菌σA 蛋白1.2次區域三個鹼性胺基酸可能的結構特性 29 圖四、枯草桿菌σA蛋白純化 30 圖五、突變型σA蛋白之純化 31 圖六、純化B. subtilis RNA聚合酶核心酵素 33 圖七、重組RNA聚合酶的體外轉錄活性(in vitro transcription activity)分析35 圖八、枯草桿菌野生型和突變型σA-RNAP與spo0A基因啟動子DNA結合能力之分析 36 附錄 37 附錄ㄧ、菌株 37 附錄二、質體 38 附錄三、本研究所使用之引子(primer) 39 附錄四、取代突變菌株之phenotype 40 附錄五、Primary σ factors胺基酸序列比對分析結果 41 附錄六、突變株之孢子形成百分比、細胞密度及生菌數 42 附錄七、φ29噬菌體G3b啟動子DNA序列 43 附錄八、kinB啟動子DNA序列 44 附錄九、spo0F啟動子DNA序列 45 附錄十、spo0B啟動子DNA序列 46 附錄十一、spo0A啟動子DNA序列 47 附錄十二、spo0E啟動子DNA序列 48 參考文獻 49zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2016-07-30起公開。zh_TW
dc.subject枯草桿菌zh_TW
dc.subjectsigma-A蛋白zh_TW
dc.subject體外轉錄實驗zh_TW
dc.subjectEMSAzh_TW
dc.subjectBacillus subtilisen_US
dc.subjectsigma-A factoren_US
dc.subjectin vitro transcriptionen_US
dc.subjectElectrophoretic Mobility Shift Assay (EMSA)en_US
dc.title枯草桿菌sigma-A蛋白N端1.2次區域三個保留性鹼性胺基酸之取代突變造成細胞提早裂解原因之探討zh_TW
dc.titleStudy of the reason responsible for the early cell lysis caused by substitutions for the three basic amino acid residues in subregion 1.2 of Bacillus subtilis σA factoren_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2016-07-30zh_TW
dc.date.openaccess2016-07-30-
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