Dimerization properties of TraM, the antiactivator that modulates TraR-mediated quorum-dependent expression of the Ti plasmid tra genes.
TraM, an 11.2 kDa antiactivator, modulates the acyl-homoserine lactone-mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum-sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the lambda cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS-PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide-bridging in TraM, altering Cys-71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild-type levels in vivo. Analysis of N-terminal, C-terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR-DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C-terminus and beginning at between residues 10 and 20 at the N-terminus play a role in TraR binding and antiactivator function. When co-expressed with lambda cI'::TraR fusions, wild-type TraM mediated quormone-independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.
Qin
Yinping
Y
Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Smyth
Audra J
AJ
Su
Shengchang
S
Farrand
Stephen K
SK
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D036741:Plant Tumor-Inducing Plasmids / Q000235:genetics* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004220:Disulfides; D011993:Recombinant Fusion Proteins; C080512:TraR protein, Agrobacterium tumefaciens; C074281:TraM protein, bacterial; C088386:homoserine lactone; D015107:4-Butyrolactone; D004229:Dithiothreitol