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J. Biol. Chem., Vol. 282, Issue 30, 22033-22039, July 27, 2007

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Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase Subunit^*

Anastasiya Sevostyanova¹, Andrey Feklistov², Nataliya Barinova, Ewa Heyduk, Irina Bass, Saulius Klimasauskas^¶, Tomasz Heyduk, and Andrey Kulbachinskiy³

From the Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq., 2, Moscow 123182, Russia, the Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, 63104, and the ^¶Institute of Biotechnology, Vilnius 02241, Lithuania

Bacterial RNA polymerase holoenzyme relies on its subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free , the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with region 1 playing a role in the masking. To analyze the DNA-binding properties of the free , we selected single-stranded DNA aptamers that are specific to primary 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 region 2 occurs without major structural rearrangements of observed upon the holoenzyme formation and is not inhibited by 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 subunit and a short aptamer oligonucleotide.

Received for publication, March 22, 2007 , and in revised form, May 25, 2007.

^* This work was supported in part by the Russian Foundation for Basic Research (Grants 05-04-49405 and 07-04-00247), by National Institutes of Health Grant GM50514 (to T. H.), by the President of the Russian Federation (Grant MK-952.2005.4 to A. K.), and by an international research fellowship grant from the Howard Hughes Medical Institute (to S. K.). Colloberation between the Institute of Biotechnology in Vilnius was supported by a travel grant from the U.S. National Academy of Sciences with partial support from the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Microbiology, The Ohio State University, Columbus, OH 43210.

² Present address: The Rockefeller University, New York, NY 10021.

¹ To whom correspondence should be addressed: Institute of Molecular Genetics, Russian Academy of Sciences, 2 Kurchatov sq., Moscow 123182, Russia. Tel./Fax: 7(499)1960015; E-mail: akulb{at}img.ras.ru.

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