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J. Biol. Chem., Vol. 282, Issue 6, 3442-3449, February 9, 2007

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Probing the Importance of Selected Phylum-specific Amino Acids in ^A of Bacteroides fragilis, a Primary Factor Naturally Devoid of an N-terminal Acidic Region 1.1^*

Didier Vingadassalom¹, Annie Kolb^¶2, Claudine Mayer, Ekkehard Collatz, and Isabelle Podglajen^||**

From the Université Paris 6 and INSERM U655-Laboratoire de Recherche Moléculaire sur les Antibiotiques, Paris F-75006, France, ^¶Unité des Régulations Transcriptionnelles, CNRS URA 2172, Institut Pasteur, Paris F-75724, France, ^||Facultéde Médecine, Université René Descartes, Paris F-75006, France, and ^**Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris F-75015, France

The ^A factor of Bacteroides fragilis is the prototype of a novel subgroup of primary factors that are essential for growth and ensure the initiation of transcription of the housekeeping genes. This subgroup is confined to the phyla Bacteroidetes and Chlorobi. Its members carry a specific amino acid signature and are notably characterized by a short, basic N-terminal segment instead of the typical acidic region 1.1. Using in vitro mutagenesis, we investigated the importance of this basic segment and of several residues of the signature for the function of ^A. We have shown that the conserved residues Phe-61 and Lys-265, located in the core binding and DNA binding subregions 2.1 and 4.2, respectively, are critical for full function of the B. fragilis holoenzyme. With respect to the unusual subregion composition of ^A, we have shown that truncation of the basic N-terminal segment, or reversion of its charge, strongly affects the overall transcriptional activity of B. fragilis RNA polymerase in vitro. Our results indicate that the presence of the intact basic segment is required for the formation of RNA polymerase (RNAP)-promoter open complexes, the correct architecture of the transcription bubble, and efficient promoter clearance.

Received for publication, September 14, 2006 , and in revised form, December 1, 2006.

^* 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 Figs. S1–S3 and Table S1.

¹ Recipient of fellowships from the Ministère de la Recherche and the Fondation pour la Recherche Médicale, Paris, France.

² To whom correspondence should be addressed: 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-45-68-86-44; Fax: 33-1-45-68-89-60; E-mail: akolb{at}pasteur.fr.

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