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J. Biol. Chem., Vol. 282, Issue 26, 19020-19028, June 29, 2007

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Direct Versus Limited-step Reconstitution Reveals Key Features of an RNA Hairpin-stabilized Paused Transcription Complex^*

Scotty Kyzer¹, Kook Sun Ha, Robert Landick², and Murali Palangat³

From the Departments of Biomolecular Chemistry and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9–10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.

Received for publication, February 20, 2007 , and in revised form, May 10, 2007.

^* This work is supported by National Institutes of Health Grant GM38660 (to R. L.). 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 Supported in part by NIGMS, National Institutes of Health, National Research Service Award T32 GM07215. Present address: Epic Systems, 1979 Milky Way, Verona, WI 53593.

² To whom correspondence may be addressed: Dept. of Biochemistry, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-265-8475; Fax: 608-262-9865; E-mail: landick{at}bact.wisc.edu. ³ To whom correspondence may be addressed: Dept. of Biochemistry, 420 Henry Mall, University of Wisconsin, Madison, WI 53706. Tel.: 608-265-8709; Fax: 608-262-9865; E-mail: palangat{at}facstaff.wisc.edu.

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