Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions
Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions
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DOI：
10.1016/j.pep.2009.09.022
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发表时间：
2010-02-01
影响因子：
1.6
通讯作者：
Nagaraja, Valakunja
中科院分区：
文献类型：
--
作者：
China, Arnab;Nagaraja, Valakunja
体外转录分析是了解转录机制的重要手段。用于分析引发、延伸和终止的各种分析方法构成了更好地理解该过程的基础。高比活性的纯化RNA聚合酶(RNAP)是进行各种特异性反应所必需的。从耻垢分枝杆菌对数期纯化的RNAP在启动子-聚合酶相互作用研究中显示出较低的启动子特异性。这是由于在指数阶段存在大量的西格玛因子，以及内务转录所需的西格玛(A)的表达不足。我们描述了RNAP全酶与Sigma(A)的体内重组及其纯化，得到了化学计量的Sigma(A)含量的全酶。与用标准方法分离的酶相比，重组全酶显示出更强的启动子特异性结合和启动子特异性转录活性。这种化学计量全酶的体内重组可以促进启动子特异性转录分析，特别是在编码大量西格玛因子的生物体中。(C)2009 Elsevier Inc.保留所有权利。
In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors. (C) 2009 Elsevier Inc. All rights reserved.