臺灣博碩士論文加值系統

在原核生物基因轉錄系統中，σ因子參與RNA聚合酶專一性辨識啟動子DNA、解開雙股啟動子DNA、早產轉錄及RNA聚合酶脫離啟動子DNA的過程。過去實驗室的研究發現，枯草桿菌N-端刪除100個胺基酸的σA蛋白 (SND100-σA)及負責環境壓力調控且缺少次區域1的σB蛋白，皆能在沒有RNA聚合酶核心酵素的協助下與啟動子DNA進行專一性的結合。進一步的研究更發現，藉由不同的蛋白重新摺疊 (refolding)方式，我們能夠得到具有不同功能特性的SND100-σA蛋白。其中，一種 (SND100-σA1)僅具有和啟動子DNA專一性結合的能力；另一種 (SND100-σA3)則還具有將啟動子雙股DNA解旋的能力。本研究想探討枯草桿菌σB蛋白是否也能夠藉由體外摺疊條件的不同，形成不同構型及功能的σB蛋白。本研究以含有sigB基因質體的E. coli BL21 (DE3)菌株，大量生產不可溶的σB蛋白，進一步利用6 M guanidine-HCl將σB蛋白聚結體變性，再以緩慢稀釋guanidine-HCl的方式，讓σB蛋白進行重新體外摺疊 (refolding)。最後，我利用以Superdex G-200以及Superdex G-75分子篩管柱層析將σB蛋白加以純化。在本實驗中，σB蛋白之重新摺疊及純化過程所用的buffer分成兩種，其中一種含有phenylmethylsulfonyl fluoride (PMSF)及Dithiothreitol (DTT)；另外一種則不含PMSF及DTT。經此純化的σB蛋白分別命名為σB1及σB3蛋白。Circular Dichroism (CD)分析結果顯示，雖然σB1和σB3之蛋白二級構造成分含率並沒有明顯差異，但整體構型卻有所不同。接著，利用electrophoretic mobility shift assay (EMSA)分析，確認σB1及σB3均具有與啟動子DNA結合能力，但和σB1蛋白相比，σB3蛋白需要較大濃度才能與啟動子DNA結合。另外，為了更進一步瞭解σB1及σB3蛋白功能上的不同，我也分析heparin對σB1及σB3與啟動子DNA複合體形成的影響。結果顯示，要抑制σB1及σB3與啟動子DNA形成複合體，所需的heparin濃度是不同的。上述結果顯示，σB1及σB3不僅在構型上不同，在DNA結合特性上也有所差異。

Transcription initiation is the first step of gene expression and a major point for gene regulation.σ factor of RNA polymerase (RNAP；α2ββ''σ) confers the specificity of promoter recognition and initiates transcription. It is also involved in open complex, abortive transcription and regulation of gene expression.
Recent works in our lab demonstrated that the N-terminally truncated primary σA factor, SND100-σA, and σB of Bacillus subtilis, both of which lack region 1.1, are able to specifically interact with promoter DNA in the absence of core RNA polymerase in vitro. Moreover, SND100-σA factors with different functional properties were achieved through the use of slightly different refolding protocols. Among them, SND100-σA1 is capable of exhibiting specific and core RNAP-independent promoter-binding activity; whereas SND100-σA3 is able to melt DNA independent of core RNA polymerase in vitro.
Present study is aimed to investigate whether the B. subtilis σB can also adopt different functional properties through protein refolding. To fulfill this goal, the σB protein was overexpressed in E. coli BL21 (DE3) harboring a σB-expressing plasmid. The overexpressed σB in inclusion bodies were denatured with guanidine hydrochloride and refolded through mild serial dilution of the guanidine hydrochloride solution. The refolded σB were soluble and can be purified using Superdex G-200 and Superdex G-75 columns. Buffers with or without both PMSF and DTT were used for protein refolding and purification and the σB proteins thus obtained were named σB1 and σB3, respectively.
Results of circular dichroism reveal that σB1 and σB3 have similar contents of secondary structures albeit with different overall conformations. Moreover, both σB1 and σB3 are able to bind promoter DNA as analyzed by electrophoretic mobility shift assay (EMSA). However, the two σB-promoter DNA complexes are different in sensitivity to heparin challenge, suggesting that they possesss different DNA-binding properties.

前言 1
材料與方法 11
一、實驗材料 11
二、菌株、質體與DNA引子來源 11
三、實驗方法 11
（一）pOB2質體之構築 11
（二）pSIGBP質體之構築 11
（三）枯草桿菌σB蛋白之大量表現 12
（四）枯草桿菌σB蛋白重新摺疊成σB3之構型及純化 12
（五）蛋白質之定量 14
（六）枯草桿菌σB1及σB3蛋白二級構造成分含率及整體構型之分析 14
（七）枯草桿菌sigB操縱組啟動子DNA之製備、純化及標示 14
（八) 枯草桿菌σB1和σB3蛋白與sigB操縱組啟動子DNA結合能力之分析 15
（九）Heparin對σB1和σB3蛋白與sigB操縱組啟動子DNA複合體形成之影響分析 15
結果 17
一、枯草桿菌σB蛋白之大量表現 17
二、枯草桿菌σB蛋白重新摺疊成σB3之構型及純化 17
三、枯草桿菌σB1和σB3蛋白二級構造成分含率及整體構型之分析 18
四、枯草桿菌σB1和σB3蛋白與sigB操縱組啟動子DNA結合能力之分析 18
五、Heparin對σB1和σB3蛋白與sigB操縱組啟動子DNA複合體形成之影響分析 19
討論 21
附圖 23
圖一、pOB2質體之構築 23
圖二、pSIGBP質體之構築 24
圖三、枯草桿菌σB蛋白之大量表現 25
圖四、枯草桿菌σB3蛋白之純化 26
圖五、枯草桿菌σB1和σB3蛋白二級構造成分含率及整體構型之分析 27
圖六、枯草桿菌σB1和σB3蛋白二級構造成分含率及整體構型之分析 29
圖七、枯草桿菌σB1和σB3蛋白與sigB操縱組啟動子DNA結合能力之分析 31
圖八、Heparin對σB1和σB3蛋白與sigB操縱組啟動子DNA複合體形成之影響分析 33
附錄一、菌株 35
附錄二、質體 36
附錄三、Primers for PCR 37
附錄四、sigB操縱組啟動子DNA序列DNA 序列 38
參考文獻 39

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