Mutational analysis of TraR. Correlating function with molecular structure of a quorum-sensing transcriptional activator.
TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to lambda cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in alpha-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.
Luo
Zhao-Qing
ZQ
Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Smyth
Audra J
AJ
Gao
Ping
P
Qin
Yinping
Y
Farrand
Stephen K
SK
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000502:physiology*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites; D004252:DNA Mutational Analysis; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; C080512:TraR protein, Agrobacterium tumefaciens; D015534:Trans-Activators; D014157:Transcription Factors