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J. Biol. Chem., Vol. 279, Issue 53, 55255-55261, December 31, 2004

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Substitutions in Region 2.4 of ⁷⁰ Allow Recognition of the ^S-Dependent aidB Promoter^*

Stephan Lacour, Olivier Leroy, Annie Kolb, and Paolo Landini¶||

From the Swiss Federal Institute of Environmental Technology (EAWAG), Überlandstrasse 133, CH-8600 Dübendorf, Switzerland, Institut Pasteur, Unité des Régulations Transcriptionnelles, Département de Microbiologie Fondamentale et Médicale (URA 1773 du CNRS), 75724 Paris, Cedex 15, France, and the ^¶Department of Molecular Biosciences and Biotechnology, University of Milan, Via Celoria 26, 20100 Milan, Italy

The strict dependence of transcription from the aidB promoter (PaidB) on the E^S form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the –10 promoter sequence (–12C), which does not allow an open complex formation by E⁷⁰. In this report, ⁷⁰ mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow –12C recognition by ⁷⁰, were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.4 of ⁷⁰ and correspond to Gln-152 and Glu-155 in ^S. Interestingly, the Q437H mutant of ⁷⁰, but not T440I, was able to promote an open complex formation and to initiate transcription at PaidB. In contrast to T440I, a T440E mutant was proficient in carrying out transcription from PaidB. No ⁷⁰ mutant displayed significantly increased interaction with a PaidB mutant in which the –12C was substituted by a T (PaidB_(C12T)), which is also efficiently recognized by wild type ⁷⁰. The effect of the T440E mutation suggests that the corresponding Glu-155 residue in ^S might be involved in –12C recognition. However, substitution to alanine of the Glu-155 residue, as well as of Gln-152, in the ^S protein did not significantly affect E^S interaction with PaidB. Our results reiterate the importance of the –12C residue for ^S-specific promoter recognition and strongly suggest that interaction with the –10 sequence and open complex formation are carried out by different determinants in the two factors.

Received for publication, September 21, 2004

^* This work was supported by Swiss National Foundation for Scientific Research Grant 3100-056742.99 (to P. L.) and a Germaine de Staël program for Swiss-French collaboration in scientific research. 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.

^|| To whom correspondence should be addressed: Dept. of Molecular Biosciences and Biotechnology, University of Milan, Via Celoria 26, 20133 Milan, Italy. Tel.: 39-02-50315028; Fax: 39-02-50315044; E-mail: paolo.landini{at}unimi.it.

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