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J. Biol. Chem., Vol. 279, Issue 32, 33093-33103, August 6, 2004

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The N Terminus of Myxococcus xanthus CarA Repressor Is an Autonomously Folding Domain That Mediates Physical and Functional Interactions with Both Operator DNA and Antirepressor Protein^*

Mari Cruz Pérez-Marín, Jose Juan López-Rubio¶, Francisco Jose Murillo, Montserrat Elías-Arnanz||, and S. Padmanabhan**

From the Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Murcia 30071, Spain

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

Received for publication, May 11, 2004 , and in revised form, May 24, 2004.

^* This work was supported by Ministerio de Ciencia y Tecnología, Spain, Grants BMC2003-00658 (to F. J. M.) and BMC2002-00539 and Programa Ramón y Cajal (to 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.

Supported by a fellowship from the Ministerio de Educación, Cultura y Deportes (Spain).

These authors contributed equally to this work.

^¶ Supported by a fellowship from Fundación Séneca (Murcia-Spain).

^|| To whom correspondence may be addressed. Tel.: 34-968-367-134; E-mail: melias{at}um.es.

^** To whom correspondence may be addressed. Tel.: 34-968-398-275; E-mail: padhu{at}um.es.

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