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J. Biol. Chem., Vol. 281, Issue 22, 15238-15248, June 2, 2006

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Antagonistic Regulation of Escherichia coli Ribosomal RNA rrnB P1 Promoter Activity by GreA and DksA^*

Katarzyna Potrykus¹, Daniel Vinella, Helen Murphy, Agnieszka Szalewska-Palasz², Richard D'Ari, and Michael Cashel³

From the Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2785 and the Institut Jacques Monod, CNRS, Université Paris 6, Université Paris 7, 7251, Paris, Cedex05, France

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

Received for publication, February 16, 2006 , and in revised form, March 31, 2006.

^* This work was supported in part by the Intramural Research Program of the National Institutes of Health, NICHD. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table SI and Figs. S1 and S2.

¹ On leave from the Dept. of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.

² Current address: Dept. of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.

³ To whom correspondence should be addressed. Tel.: 301-496-0619; Fax: 301-496-0243; E-mail: mcashel{at}nih.gov.

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