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J. Biol. Chem., Vol. 280, Issue 50, 41315-41323, December 16, 2005

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RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

CRITICAL NUCLEOTIDES IN THE -7 TO-18 REGION ARE NEEDED TO SELECT BETWEEN RNA POLYMERASE WITH ³⁸ OR ^32*

From the Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, E-18008 Granada, Spain

The Pm promoter of the benzoate meta-cleavage pathway is transcribed with E³² or E³⁸ according to the growth phase, with an identical transcriptional start site. To investigate sequence determinants in the interaction between either of the two RNA polymerases and Pm, all possible single mutants between positions -7 and -18 were generated, and the activity in the exponential and stationary phases of the resulting mutant promoters was compared. The results precisely delimited a -10 element between positions -7 and -12 (TAGGCT), which defined a promoter sharing nucleotides with both ³⁸ and ³² consensus. The first two and the last positions of this hexamer were crucial for recognition by both polymerases. Position -10 was the only one specifically recognized by E³⁸, whereas positions -8, -9, and the C-track between positions -14 and -17 were important for specific E³² recognition. Western blots showed that ³² was only detectable in the exponential phase, and ³⁸ appeared in the early stationary phase. In the rpoH mutant KY1429, ³⁸ was already present in the exponential growth phase both free and bound to the RNA polymerase core, in good correlation with the transcription levels found. Pm seems to be optimized for recognition by ³² as an initial response to the addition of effector to the medium and allows binding of the adaptable ³⁸-dependent RNA polymerase in the stationary phase. XylS is always required for Pm transcription. Therefore, the mechanism that controls Pm expression involves specific nucleotide sequences, the abundance of free and core-bound ³² and ³⁸ factors during growth, and the presence of the regulator activated by an effector.

Received for publication, May 17, 2005 , and in revised form, September 28, 2005.

^* This work was supported by Grants BMC2001-0515 and QLK3-2002-01923 from the Spanish Ministry of Science and Technology (MCYT). 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 an I3P contract from the European Social Funds and a fellowship from the Junta de Andalucía (Andalusian Regional Government, Spain).

² To whom correspondence should be addressed: Estación Experimental del Zaidín, CSIC, C/o Profesor Albareda 1, E-18008 Granada, Spain. Tel.: 34-958-181600; Fax 34-958-129600; E-mail: silvia{at}eez.csic.es.

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