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J. Biol. Chem., Vol. 282, Issue 27, 19979-19991, July 6, 2007

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Molecular Basis of Transcriptional Antiactivation

TraM DISRUPTS THE TraR-DNA COMPLEX THROUGH STEPWISE INTERACTIONS^*

From the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Conjugative transfer of Agrobacterium Ti plasmids is regulated by TraR, a quorum-sensing activator. Quorum dependence requires TraM, which binds to and inactivates TraR. In this study, we showed that TraR and TraM form a 151-kDa stable complex composed of two TraR and two TraM dimers both in vitro and in vivo. When interacted with TraR bound to tra box DNA, wild-type TraM formed a nucleoprotein complex of 77 kDa composed of one dimer of each protein and DNA. The complex converted to the 151-kDa species with concomitant release of DNA with a half-life of 1.6 h. TraR in the complex still retained tightly bound autoinducer. From these results, we conclude that TraM interacts in a two-step process with DNA-TraR to form a large, stable antiactivation complex. Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex. From these genetic and structural considerations, we constructed three-dimensional models of the complex that shed light on the mechanism of TraM-mediated inhibition of TraR and on TraM-mediated destabilization of the TraR-DNA complex.

Received for publication, April 20, 2007

^* This work was supported in part by National Institutes of Health Grant R01GM52465 (to S. K. F.). 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 To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois at Urbana-Champaign B103 CLSL, 601 S Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1524; Fax: 217-244-6697; E-mail: stephenf{at}uiuc.edu.

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