TrlR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrlR:TraR dimers.
Octopine-type Ti plasmids of Agrobacterium tumefaciens require the quorum-sensing proteins TraR and TraI and the diffusible pheromone 3-oxooctanoyl homoserine lactone (AAI) to regulate genes required for conjugal transfer. TraR activity is inhibited by a protein called TrlR, which closely resembles amino acids 1-181 of TraR but is truncated as a result of a shift in the reading frame at codon 182. This frameshift does not affect synthesis of the amino-terminal domain, which is thought to bind autoinducer and mediate protein dimerization, but abolishes translation of the carboxyl-terminal, DNA-binding domain. In this study, we show that TrlR, like TraR, requires AAI for solubility when overexpressed in Escherichia coli. TrlR bound one molecule of AAI per protein monomer, supporting the prediction that the amino-terminal domain of TraR contains the AAI binding site. Purified TrlR blocked TraR for both specific DNA binding and transcription of a tra promoter, supporting previous studies performed with whole cells. When TrlR and a TraR fusion protein were co-expressed in E. coli, these proteins readily formed heterodimeric complexes that were inactive in DNA-binding activity. These data support the hypotheses that (i) the amino-terminal half of TraR binds AAI and mediates protein dimerization; (ii) both DNA-binding domains in a TraR dimer are required for stable DNA binding; and (iii) TrlR blocks TraR by direct protein-protein interactions.
Chai
Y
Y
Department of Microbiology, 360A Wing Hall, Cornell University, Ithaca, NY 4853, USA.
Zhu
J
J
Winans
S C
SC
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C080512:TraR protein, Agrobacterium tumefaciens; C434270:TrlR protein, Agrobacterium tumefaciens; C088386:homoserine lactone; D015107:4-Butyrolactone