Sevostyanova Anastasiya, Feklistov Andrey, Barinova Nataliya, Heyduk Ewa, Bass Irina, Klimasauskas Saulius, Heyduk Tomasz, Kulbachinskiy Andrey
Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq, 2, Moscow, Russia.
J Biol Chem. 2007 Jul 27;282(30):22033-9. doi: 10.1074/jbc.M702495200. Epub 2007 May 29.
Bacterial RNA polymerase holoenzyme relies on its sigma subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the sigma subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free sigma, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with sigma region 1 playing a role in the masking. To analyze the DNA-binding properties of the free sigma, we selected single-stranded DNA aptamers that are specific to primary sigma subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by sigma region 2 occurs without major structural rearrangements of sigma observed upon the holoenzyme formation and is not inhibited by sigma regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated sigma subunit and a short aptamer oligonucleotide.
细菌RNA聚合酶全酶依靠其σ亚基来识别和打开启动子。在全酶中，σ亚基的2区和4区以最佳距离定位，分别允许特异性识别-10和-35启动子元件。在游离的σ中，启动子结合区域彼此靠得更近，并且与启动子的相互作用被掩盖，其中σ1区在这种掩盖中起作用。为了分析游离σ的DNA结合特性，我们选择了对几种细菌物种（包括大肠杆菌和嗜热水生栖热菌）的主要σ亚基具有特异性的单链DNA适体。这些适体共享一个共有基序TGTAGAAT，它类似于扩展的-10启动子。我们证明，σ2区对该基序的识别在全酶形成时未观察到σ的主要结构重排的情况下发生，并且不受σ1区和4区的抑制。因此，RNA聚合酶全酶识别-10元件的复杂过程可以简化为一个由分离的σ亚基和短适体寡核苷酸组成的简单系统。
