Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92195
標題: 在缺乏核心酵素下主要的sigma因子與啟動子DNA專一性能力之探討
Study on the core-independent promoter-specific interaction of primary sigma factors
作者: 林子峰
Tzu-Feng Lin
關鍵字: sigma因子;sigma factor
引用: 1. Aiyar, S.E., Gourse, R.L., and Ross, W. (1998). Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit. Proc Natl Acad Sci U S A 95, 14652-14657. 2. Amaya, E., Khvorova, A., and Piggot, P.J. (2001). Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides. J Bacteriol 183, 3623-3630. 3. Arthur, T.M., Anthony, L.C., and Burgess, R.R. (2000). Mutational analysis of beta' 260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase. J Biol Chem 275, 23113-23119. 4. Arndt, K.M., and Chamberlin, M.J. (1988). Transcription termination in Escherichia coli. Measurement of the rate of enzyme release from Rho-independent terminators. J. Mol. Biol. 202, 271-285. 5. Artsimovitch, I. (2008). Post-initiation control by the initiation factor sigma. Mol Microbiol 68, 1-3. 6. Badaut, Cyril., Williams, Roy., Arluison, Ve'ronique., Bouffartigues, Emeline., Robert, Bruno., Buc, Henri., Rimsky, Sylvie (2002). The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA. J Biol Chem 277, 41657-41666. 7. Blatter, E.E., Ross, W., Tang, H., Gourse, R.L., and Ebright, R.H. (1994). Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell 78, 889-896. 8. Bloch V, Yang Y, Margeat E, Chavanieu A, Auge MT, Robert B, Arold S, Rimsky S, Kochoyan M. (2003). The H-NS dimerization domain defines a new fold contributing to DNA recognition. Nat Struct Biol. 10, 212-218 9. Bochkareva, A., Zenkin, N. (2013). The
摘要: 
原核生物轉錄起始因子σ,負責協助RNA聚合酶 (RNA polymerase)專一性的辨識啟動子DNA,並引發基因之轉錄。不過,σ是否能夠像真核細胞的轉錄起始因子一樣,直接與啟動子DNA結合,再招募核心酵素 (core enzyme),來啟動基因轉錄,卻一直沒有定論。實驗室利用蛋白質體外重新摺疊、及管柱純化的方法,製備了枯草桿菌主要的σ (σA)蛋白樣品,並利用Electrophoretic Mobility Shift Assay (EMSA)及Footprinting等技術,發現這個樣品在體外 (in vitro)的情況下,不需要核心酵素的協助,即可與啟動子-10 DNA進行專一性結合;另外,實驗室學長也利用Sequential Chromatin Immunoprecipitation (SeqChIP)等技術,偵測到在枯草桿菌體內的σA蛋白也能與啟動子DNA進行專一性結合。不過這種菌體內σA與啟動子DNA結合的訊號,雖可能來自於σA直接與啟動子DNA的結合,但也有可能來自於RNA聚合酶與啟動子DNA結合後,核心酵素離開σA與啟動子DNA複合體所形成的。為了排除後者因素,我必需得到核心酵素的次單元體具有突變的溫度敏感菌株。文獻記載,大腸桿菌RNA聚合酶α因子第45及191之Arg胺基酸以Cys取代,會導致RNA聚合酶在高溫下產生組裝上之缺陷或造成β及β′因子被分解。利用這種資訊,我將枯草桿菌α因子和大腸桿菌α因子同源第42及184胺基酸位置上的Arg進行Cys取代突變,希望能找到RNA聚合酶組裝缺陷的溫度敏感突變菌株。為達成這個目地,首先我以含有刪除核醣體結合位置及蛋白N端8個胺基酸密碼的α基因質體,送進枯草桿菌中進行DNA的重組,篩選α因子第42、184位置具有單點或雙點突變的枯草桿菌,但在利用四環素 (tetracycline)篩選時,雖可篩選到對溫度敏感的菌株,但α基因的目標位置上,並沒有發生突變的現象,推測此可能和 tetracycline造成枯草桿菌的生長遲滯有關。為了解決此生長遲滯的問題,我進一步將送入枯草桿菌中含有α基因突變質體上四環素抗菌基因以大觀霉素 (spectinomycin)抗菌基因取代,再進行DNA重組,雖發現枯草桿菌已無生長遲滯的狀況。但在分別篩選了約八百株轉型細胞之後,卻只各得到一株具有溫度敏感的突變株,不過此菌株染色體上之α基因經DNA定序後,目標位置上並沒有Arg-to-Cys取代突變發生。根據上述研究結果,我推測無法獲得α基因突變的溫度敏感突變株的原因,可能和α基因的突變造成枯草桿菌的致命性有關。

The prokaryotic transcription factor, σ, is responsible for promoter specific recognition by RNA polymerase and is able to initiate transcription. However, whether primary σ could independently bind to the promoter DNA similar to other transcription factors remains unclear. Recently, our lab found that an in vitro refolded Bacillus subtilis σA sample is able to specifically interact with promoter DNA in a core-independent manner. In addition, specific interaction between σ70 and its cognate promoter DNA was also detected in Bacillus subtilis using Sequential Chromatin Immunoprecipitation (SeqChIP) assay. This study is aimed to confirm the latter finding by excluding the possibility that the in vivo binding signal was obtained indirectly through dissociation of core RNA polymerase from the holoenzyme and promoter DNA binary complex. In order to fulfill this goal, I have to get a temperature-sensitive (Ts) core mutant of B. subtilis. And the α subunit of core RNA polymerase is our choice. It has been reported that the replacement of Arg-45 or Arg-191 with cysteine in Escherichia coli RNA polymeraseαsubunit would lead to a Ts phenotype of E. coli. In this study, Arg-to-Cys substitution at corresponding positions (Arg-42 or Arg-184) of the B. subtilis rpoA gene was created and cloned into an integration vector after deletion of the ribosome binding site and the first 8 amino acid codons for rpoA. Transformation of the integration plasmid into B. subtilis would allow integration of the mutated rpoA into the locus of endogenous rpoA by single crossing-over event. The results showed that the growth of all the integrants was significantly retarded when tetracycline was incorporated into the medium. Moreover, no rpoA mutant with Arg-42-Cys or Arg-184-Cys substitution was obtained. To solve the problem of growth retardation of B. subtilis by tetracycline, I replaced the tetracycline resistance gene with the spectinomycin resistance gene on the integration plasmid and performed the same integration experiment. The results showed that the growth of the integrants was no longer retarded and only one integrant out of the 800 obtained for each of the designed rpoA mutation was Ts. Unfortunately, the rpoA sequence on the chromosome remained unchanged for the Ts cells. These results lead me to propose that the Arg-42-Cys or Arg-184-Cys substitution in B. subtilisαsubunit is lethal to the cells.
URI: http://hdl.handle.net/11455/92195
Rights: 同意授權瀏覽/列印電子全文服務,2017-01-16起公開。
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