Identification of amino acid residues of the pheromone-binding domain of the transcription factor TraR that are required for positive control

Abstract

Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promoter, where TraR binds to a site centred 45 nucleotides upstream of the transcription start site, and of the traM promoter, where TraR binds a site centred 66 nucleotides upstream. Alteration of 18 residues blocked activity at the traI promoter. Of these, alteration at three positions impaired TraR abundance or DNA binding, leaving 15 residues that are specifically needed for positive control. Of these 15 residues, nine also blocked or reduced activity at the traM promoter, while six had no effect. Amino acid residues required for activation of both promoters probably contact the C-terminal domain of the RNA polymerase alpha subunit, while residues required only for traI promoter activation may contact another RNA polymerase component.

Figure 1

Saturation mutagenesis of the surface of the NTD of TraR. Residues in white were altered by site-directed mutagenesis.

Figure 2

Western immunoblot data of TraR point mutants in A. tumefaciens strain NTL4 (pCEW260). Strains containing pYC335 or pPZP201served as positive and negative controls, respectively. Purified TraR was also used as a positive control for all westerns (left lane). The cross-reacting material (CRM) was used to normalize the intensity of each TraR band.

Figure 3

Electrophoretic mobility shift assays with TraR in crude cell extracts. The amount of full-length, soluble TraR in each extract was normalized using western immunoblots.

Figure 4

Intragenic complementation of mutations of D10, G123, and W184. Successful complementation was taken as an indication that the amino acid residues are contributed by opposite subunits.

Figure 5

Positive control mutants isolated in this study (in the TraR NTD) and in a previous study (TraR-CTD) (White and Winans, 2005). Residues in white affect both PtraI and PtraM, while residues in black affected only PtraI. (A) View of TraR along the DNA axis, showing the concave and convex faces of the crystal structure. (B and C) TraR rotated to show the concave surface and convex surfaces, respectively.

Figure 6

Closeup views of TraR residues required for positive control. Residues in white are needed for both promoters, while residues in black are needed only for PtraI. (A) A patch on the concave surface, composed of residues from both subunits and both domains of each subunit. (B) A patch on the convex surface, composed of residues from the NTDs of both subunits.