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dc.description.abstract細菌細胞中之轉錄起始因子,σ,能否如同真核生物之轉錄因子一般,先與啟動子 DNA 結合,再招攬 RNA 聚合酶,來啟動轉錄的進行,一直是生命科學家們想解答的問題。因此,純化具有直接與啟動子 DNA 結合能力的蛋白,並在細胞中偵測到這種結合的存在,就成為解答這個問題的重點。本研究中,我發現必需採用較緩慢的體外重新摺疊,才能由不可溶的σA 蛋白聚結體(inclusion bodies)樣品中,製備具有獨立與啟動子 DNA 結合能力的σA 蛋白。不過,體外重新摺疊所獲得的σA 樣品,存在有不同構形及和啟動子 DNA 不同結合能力的σA 蛋白,必需再利用分子篩層析管柱,加以純化。此經重新摺疊、純化的σA 蛋白與啟動子 DNA 結合能力和從大腸桿菌細胞中表現、純化的自然摺疊、水溶性σA 蛋白相似,可見不需要核心酵素(core enzyme)的協助,σA 蛋白即具有與啟動子 DNA 結合之本能力。為了證明枯草桿菌細胞內之A 蛋白也具有獨立與啟動子 DNA 結合之能力 我進行了,Sequential Chromatin Immunoprecipitation (SeqChIP)的分析。結果顯示,在枯草桿菌指數生長時期中,σA 蛋白可以直接與 ezrA、sigA-P1P2、veg、spo0A、spo0H 和29噬菌體 G3b 等啟動子 DNA 結合,而且至少與 G3b 啟動子 DNA 之–10 及–35 elements 的結合具有專一性。另外,σA 蛋白也具有辨識最佳啟動子–10 及–35 elements 之區間(spacing)的能力 因子若真能在沒有核心酵素協助下即能與啟動子σDNA 結合,那麼它將帶給原核生物基因轉錄起始研究一個嶄新的方向。zh_TW
dc.description.abstractIt has been long a focus for scientists to answer whether the prokaryotic σ like the universal eukaryotic transcription initiation factor, the TATA box-binding protein, can be recruited to the promoter prior to association with core RNAP during transcription initiation. To answer this question, purification of σ with the promoter DNA-binding property in vitro and detection of the σ-promoter-DNA interaction in vivo are absolutely required. In my study, I found that the Bacillus subtilis σA with a promoter DNA-binding activity can be obtained if the σA was overexpressed heterologously in Escherichia coli, denatured, refolded slowly in vitro and purified with molecular sieving columns. The promoter DNA-binding activity of the in vitro refolded σA is similar to that overproduced in Escherichia coli in soluble form. These results support the idea that the observed promoter DNA-binding activity of the refolded σA is not an artifact but is an intrinsic property of σA. The interaction of σA with known promoter DNAs in B. subtilis was analyzed by Sequential Chromatin Immunoprecipitation (SeqChIP) in accompany with a Nickel-resin affinity chromatography. The results demonstrated that σA is at least able to interact with ezrA, sigA-P1P2, veg, spo0A, spo0H and σ phage G3b promoters in B. subtilis in early log phase. Both –10 and –35 elements of the G3b promoter DNA are required for the efficient promoter-specific interaction of σ A in vivo.Moreover, the promoter –10 and –35 specific interaction of σA is able to allow the σ A to discern the optimal promoter spacing in vivo. The ability of σ by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function and gene regulation during transcription initiation.en_US
dc.description.tableofcontentsContents Introduction ............................................. 1 Materials and Methods Overproduction and purification of σA ................... 10 Labeling of the σ phage G3b promoter DNA ................ 10 Electrophoretic mobility shift assay (EMSA) ............. 11 DNase I footprinting assay............................... 11 In vitro transcription assay ............................ 12 Sequential chromatin immunoprecipitation (SeqChIP) assay 13 Construction of B. subtilis DB430C12 .................... 14 Integration of the G3b promoter DNA into B. subtilis chromosome .............................................. 14 Construction of spacing variants of the G3b promoter DNA ......................................................... 15 Integration of each of the G3b promoter spacing variants into B. subtilis chromosome ............................. 15 Construction of temperature-sensitive mutations in the α subunit gene of B. subtilis RNA polymerase............... 16 Results Purification of the B. subtilis primary A, with a core-independent promoter DNA-binding activity ........ 18 The existence of core-independent promoter interaction of σA in B. subtilis .......................................... 19 Both –10 and –35 elements are important for core-independent promoter-specific interaction of σA in vivo ............. 19 The promoter spacing is critical for efficient formation of the σA-promoter DNA complex ............................. 20 Construction of the temperature-sensitive mutations in the α subunit gene of B. subtilis RNA polymerase .............. 21 Discussion .............................................. 22 References............................................... 39zh_TW
dc.title枯草桿菌 Sigma-A 因子和啟動子 DNA 專一性結合活性之體內分析zh_TW
dc.titleStudy on the specific promoter DNA-binding activity of Bacillus subtilis Sigma-A factor in vivoen_US
dc.typeThesis and Dissertationen_US
item.openairetypeThesis and Dissertation-
item.fulltextwith fulltext-
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