Characterization of the rpoC gene of Streptomyces coelicolor A3 (2) and its use to develop a simple and rapid method for the purification of RNA polymerase
Characterization of the rpoC gene of Streptomyces coelicolor A3 (2) and its use to develop a simple and rapid method for the purification of RNA polymerase
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DOI：
10.1016/s0378-1119(97)00179-0
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发表时间：
1997-09-01
期刊：
影响因子：
3.5
通讯作者：
Brawner, ME
中科院分区：
文献类型：
--
作者：
Babcock, MJ;Buttner, MJ;Brawner, ME
使用大肠杆菌 rpoC 基因作为杂交探针分离编码 RNA 聚合酶 β' 亚基的天蓝色链霉菌 rpoC 基因。将天蓝色链球菌β'亚基的预测氨基酸序列与其他细菌特征的氨基酸序列进行比较，揭示了β'亚基的三个不同亚科，其中一个由天蓝色链球菌亚基以及来自麻风分枝杆菌和生殖支原体的亚基组成。使用定点诱变，天蓝色链球菌β'亚基的羧基末端被修饰为包含六个组氨酸残基。组氨酸标记基因 rpoC(HIS) 用于替换 S. coelicolor 和 S. lividans 染色体中的野生型等位基因。这些菌株的生长和孢子形成未受影响，表明组氨酸标记的 RNA 聚合酶能够执行所有必要的体内功能。在 1 天的过程中，通过镍-NTA 琼脂糖亲和层析和肝素-琼脂糖层析获得高度纯化的 RNA 聚合酶。使用体外径流转录测定，亲和纯化的 RNA 聚合酶被证明可以从 S. lividans galP1 和 galP2 启动子以及枯草芽孢杆菌 veg 和 ctc 启动子正确启动转录。对该过程的扩展产生了高度纯化的核心RNA聚合酶。为了促进将 rpoC(HIS) 等位基因引入其他遗传背景，在 S, coelicolor 中分离了邻近基因 rpoB (rifA) 中赋予利福平抗性的突变，以提供跟随 rpoC(HIS) 等位基因转移的遗传标记。这种亲和层析方法的使用，与通过转化将rpoC(HIS)等位基因引入不同链霉菌菌株的能力相结合，将极大地促进该属成员转录的体外分析。 (C) 1997 Elsevier Science B.V.
The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparision of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoC(HIS), was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoC(HIS) allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S, coelicolor to provide a genetic marker to follow transfer of the rpoC(HIS) allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoC(HIS) allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus. (C) 1997 Elsevier Science B.V.