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J. Biol. Chem., Vol. 282, Issue 25, 18046-18056, June 22, 2007

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Properties of RNA Polymerase Bypass Mutants

IMPLICATIONS FOR THE ROLE OF ppGpp AND ITS CO-FACTOR DksA IN CONTROLLING TRANSCRIPTION DEPENDENT ON ^54*

Agnieszka Szalewska-Palasz, Linda U. M. Johansson, Lisandro M. D. Bernardo¹, Eleonore Skärfstad, Ewa Stec, Kristoffer Brännström, and Victoria Shingler²

From the Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden and the Department of Molecular Biology, University of Gdansk, 80822 Gdansk, Poland

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain ⁷⁰-dependent promoters. A number of promoters that are dependent on alternative -factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived ⁵⁴-dependent Po promoter and several other ⁵⁴-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro ⁵⁴-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the - and '-subunits of core RNA polymerase that allow high levels of transcription from the ⁵⁴-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with -factors to favor ⁵⁴-holoenzyme formation over that with ⁷⁰, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent ⁷⁰-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of ⁵⁴-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent ⁷⁰-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative -factors is controlled to meet cellular requirements.

Received for publication, October 31, 2006 , and in revised form, April 23, 2007.

^* This work was supported in part by grants from the Swedish Foundation for Strategic Research and the Swedish Research Council (to V. S.) and the Polish Ministry of Education and Science (Grant 2 PO4A 034 28, to A. S.-P.). 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 Recipient of a student fellowship from the Foundation for Science and Technology (FCT, Portugal).

² To whom correspondence should be addressed. Tel.: 46-90-785-2534; Fax: 46-90-771420; E-mail: victoria.shingler{at}molbiol.umu.se.

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