Please use this identifier to cite or link to this item:
標題: 枯草桿菌RNA聚合酵素sigma-A因子之構造、功能及調控之研究
Studies on the Structure, Function and Regulation of Bacillus subtilis RNA Polymerase sigma-A Factor
作者: 廖朝財
Liao, Chao-Tsai
關鍵字: sigma-A factor;sigma-A 因子;Bacillus subtilis;rpoD (sigma-A) operon;temperature-sensitive;枯草桿菌;rpoD (sigma-A) 基因組;溫度敏感
出版社: 農業生物科技學研究所
根據許多已知sigma蛋白胺基酸序列的比對分析結果,一般sigma蛋白具有四個保留性區域(conserved region),C-端的4.2區域具有一個helix-turn-helix的構造,其和起動子-35 DNA的結合有關。sigma蛋白和起動子-10 DNA結合的位置,則在於這個蛋白的2.4區域。依據Chou及Fasman氏蛋白質二級構造之分析結果,位在Bacillus subtilis sigma-A蛋白2.4區域的胺基酸,會形成一個具有親水及疏水兩面性的a-螺旋體(amphiphilic a-helix)。在這個螺旋體的疏水性面上有Ile-194, Ile-198及Ile-202等三個保留性胺基酸,一般認為這三個疏水性胺基酸在自由態sigma-A蛋白中,可能會與蛋白內其它區域的疏水性胺基酸進行疏水性結合,以穩定sigma-A蛋白的構造。為瞭解這三個保留性Ile對sigma-A蛋白構造及功能的影響,本實驗室利用基因定點突變(site-directed mutagenesis)的方式,在不改變sigma-A蛋白2.4區域a-螺旋體特性下,將疏水性的Ile變成Ala,並獲得了一株對溫度敏感(temperature-sensitive, Ts)的B. subtilis sigA突變株,命名為B. subtilis DB1005,此菌株之sA蛋白具有Ile-198-Ala及Ile-202-Ala之雙重取代。
本研究首先探討造成B. subtilis DB1005在高溫下對溫度敏感的原因。研究結果顯示,這個突變型sigma-A蛋白,不但構造不穩定,而且轉錄活性也降低。但是此菌株之對溫度敏感,並非由於突變型sigma-A蛋白之構造不穩、被分解,細胞內該蛋白含量不足所致,而是由於該蛋白之活性不足。接著,我們亦比較三個保留性Ile對sigma-A蛋白的構造、及功能重要性的差異。結果顯示,這三個Ile對sigma-A蛋白的構造及功能,具有差異性及加成性的影響力,其中Ile-198在維持sigma-A蛋白的構造穩定性及轉錄活性上,均比Ile-194與Ile-202更為重要。此外,為瞭解完整sigma-A蛋白和起動子-10 DNA結合區域之a-螺旋體結構,對sigma-A蛋白之構造及功能的重要性,我們亦構築了兩株a-螺旋體遭受破壞的B. subtilis sigA突變株。結果顯示,維持完整sigma-A蛋白和起動子-10 DNA結合區域之a-螺旋體結構,不但對維持sigma-A蛋白構造之穩定是必需的,對確保sigma-A蛋白的轉錄功能也非常重要。最後,我們探討在相同轉錄(transcription)及轉譯(translation)信號控制下,B. subtilis DB1005(pCX2F5)細胞內溫度敏感sigA基因和B. subtilis DB2(pCX2F)細胞內的野生型sigA基因,為何在高溫下會有明顯的差異性表現。我們意外發現B. subtilis的大分子合成基因組(macromolecule synthesis [MMS, rpoD or sigA] operon)中有一個未被鑑定的sigma-B type 起動子的存在,將其命名為P7起動子。 Primer extension結果顯示,當B. subtilis於熱(heat)、酒精(ethanol)及鹽休克(salt stress)下,P7起動子會受到不同程度的誘導。
整體而言,不論改變sigma-A蛋白和起動子-10 DNA結合區域之a-螺旋體的螺旋性(helicity)或疏水性(hydrophobicity),皆會導致sigma-A蛋白結構之不穩定,即使在允許細菌生長的溫度下(37℃),sigma-A蛋白亦容易被分解。同時,這些變異也會導致sigma-A蛋白於高溫(49℃)下,失去其活性。可見,sigma-A蛋白和起動子-10 DNA結合的區域,不僅參與起動子-10 DNA之辨識,也具有維持sigma-A蛋白結構穩定、功能完整的功能。研究結果也顯示,至少有七個起動子參與B. subtilis大分子合成基因組(MMS operon)表現的調控,它們可以確保在不同細胞發展期(cell development)或環境壓力(environmental stress)下,生產本基因組的蛋白產物。

Sequence alignment analyses of known sigma factors have revealed that this group of proteins contains four conserved regions. Region 4.2 at C-terminus has a helix-turn-helix structure which is believed to interact with the -35 region of promoter DNA. Region 2.4 which recognizes the promoter -10 DNA has an amphiphilic a-helix with three isoleucine residues located at four residue intervals. The three isoleucine residues were assumed to face the hydrophobic core of sigma factor and stabilize the sigma protein. In order to confirm the roles of the three conserved isoleucines, we constructed several B. subtilis sigA mutants containing either one or two Ile-to-Ala substitutions on the promoter -10 recognition helix. One of the sigA mutants (termed B. subtilis DB1005) contains double-amino-acid substitutions, I198A and I202A, and is very sensitive to temperature elevation.
In this study, I first investigated the nature of temperature sensitivity of B. subtilis DB1005. The results clearly showed that the temperature sensitivity of this sigA mutant is due to insufficient activity, rather than insufficient concentration, of the mutant sigma-A factor. Then, I clarified the contribution of each of the conserved isoleucines to sigma-A structure and function. The results indicated that the three conserved isoleucine residues (Ile-194, Ile-198 and Ile-202) are all involved in the hydrophobic core packing of sigma-A; they affect differentially and additively the structure and function of sigma-A, with the central isoleucine residue (Ile-198) playing the most important role. I also investigated the importance of maintaining a proper helical structure in the promoter -10 binding region to sigma-A structure and function. The results revealed that the maintenance of a proper helical structure in the promoter -10 binding region is not only essential to overall structural stability but also important for ensuring a normal function of sigma-A. Finally, I studied the regulation of expression of the sigA gene and localized a stress-responsive sigma-B type promoter, P7, in the B. subtilis macromolecular synthesis (MMS, rpoD or sigA) operon. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress.
On the whole, the results reveal that both the changes of helicity and hydrophobicity of the promoter -10 binding helix destabilize sigma-A, leading to its degradation even at the permissive temperature and loss of activity at the restrictive temperature. Therefore, the promoter -10 binding region is not only essential to promoter recognition but also to maintenance of a stable and functional sigma-A structure. Furthermore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the five known sigma-A, sigma-H and sigma-B type promoters and two incompletely defined promoters. These complex promoters ensure the transcription of the B. subtilis MMS operon to be kept at a certain level during cell development or environmental stress.
Appears in Collections:生物科技學研究所

Show full item record
TAIR Related Article

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.