Dimerization of the quorum-sensing transcription factor TraR enhances resistance to cytoplasmic proteolysis.
TraR is a LuxR-type quorum-sensing protein encoded by the tumour-inducing plasmid of Agrobacterium tumefaciens. TraR requires the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two alpha-helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo. Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using Western immunoblots and OOHL sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding and protein half-life. We conclude that dimerization of TraR enhances resistance to cellular proteases.
Pinto
Uelinton M
UM
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Winans
Stephen C
SC
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D053038:Quorum Sensing; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C511789:N-(3-oxooctanoyl)homoserine lactone; C080512:TraR protein, Agrobacterium tumefaciens; D014157:Transcription Factors; D015107:4-Butyrolactone