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J. Biol. Chem., Vol. 279, Issue 39, 40844-40851, September 24, 2004

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Domains Formed within the N-terminal Region of the Quorumsensing Activator TraR Are Required for Transcriptional Activation and Direct Interaction with RpoA from Agrobacterium^*

Yinping Qin, Zhao-Qing Luo, and Stephen K. Farrand¶||

From the Departments of Crop Sciences and ^¶Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters. TraR activated expression of a lacZ reporter in Escherichia coli only when RpoA_At from Agrobacterium tumefaciens was co-expressed. As assessed by gel retardation assays RpoA_At, but not RpoA_Ec, formed a ternary complex with TraR and a tra-box probe in vitro. TraR formed similar ternary complexes with CTD_At but not with NTD_At, the C- and N-terminal segments of RpoA_At. As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoA_At with an affinity about five times greater than that observed for its interaction with RpoA_Ec. The activator interacted with CTD_At with kinetics and affinities similar to those of the full-sized -subunit. Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoA_At interaction. These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA. When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner. Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function.

Received for publication, May 12, 2004 , and in revised form, June 24, 2004.

^* This work was supported in part by Grant R01 GM52465 from the National Institutes of Health (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.

Current address: Dept. of Biological Sciences, Purdue University, West Lafayette, IN 47907.

^|| To whom correspondence should be addressed: Dept. of Crop Sciences, University of Illinois at Urbana-Champaign, 240 ERML, 1201 West Gregory Dr., Urbana, IL 61801. Tel.: 217-333-1524; Fax: 217-333-4582; E-mail: stephenf{at}uiuc.edu.

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