The antiactivator TraM interferes with the autoinducer-dependent binding of TraR to DNA by interacting with the C-terminal region of the quorum-sensing activator

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoseri ne lactone Agrobacterium autoinducer (AAI). Unique to this system, the acti vity of TraR is negatively modulated by an antiactivator called TraM, Analy ses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact an d the region of TraR responsible for this interaction are not known, Induct ion of traM in a strain in which TraR was activating transcription of a rep orter system led to rapid cessation of gene expression. As assessed by a ge netic assay that measures AAI-dependent DNA binding, TraM inhibited TraR fu nction before and after the transcription factor had bound to its DNA recog nition site. Consistent with this observation, in gel retardation assays, p urified TraM abolished the DNA binding activity of TraR in a concentration- dependent manner. Such inhibition occurred independent of the order of addi tion of the reactants. As assessed by far Western analyses TraM interacts w ith TraR by directly binding the activator. TraM in its native form interac ted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein. TraM interacted with TraR on blots pre pared with total lysates of cells grown in the presence and absence of AAI. Far Western analysis of N- and C-terminal deletion mutants localized a dom ain of TraR contributing to TraM binding to the e-terminal portion of the a ctivator protein. Random mutagenesis by hydroxylamine treatment and error-p rone polymerase chain reaction identified several residues in this region o f TraR important for interacting with TraM as well as for transcriptional a ctivation or/and DNA binding. We conclude that TraM inhibits TraR by bindin g to the activator at a domain within or close to the helix-turn-helix moti f located at the C terminus of the protein.