title: Error-prone PCR mutagenesis reveals functional domains of a bacterial transcriptional activator, TraJ.
abstract: TraJ is the essential activator of P(Y), the promoter of the F and F-like plasmid tra operon that encodes the majority of the proteins for bacterial conjugation. By combining error-prone PCR mutagenesis with a two-plasmid screen, we isolated 55 missense mutations in traJ, each affecting the ability of TraJ to activate P(Y). These mutations define two distinct functional clusters (amino acids [aa] 21 to 117 and aa 150 to 219). Limited proteolytic analysis of TraJ suggested that the N- and C-terminal functional clusters are two structurally distinct domains. Most TraJ mutants exhibited decreased intracellular protein levels, and the HslVU protease-chaperone pair was found to be responsible for degrading those mutants without extracytoplasmic stress-induced overexpression. In vivo cross-linking analysis of TraJ mutants indicated that the N-terminal domain is responsible for dimerization. This was confirmed by the finding that the purified N-terminal region of TraJ forms dimers in solution. The levels of dimerization and in vivo activities of TraJ mutants are well correlated, suggesting that dimerization of TraJ is required for its biological function. We propose that the regulation of TraJ dimerization and/or its susceptibility to HslVU could be a key mechanism in various signaling processes for controlling bacterial conjugation in response to physiological or environmental stimuli.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Yun
authors.initials: Y
authors.lastname: Peng
authors.forename: Denis
authors.initials: D
authors.lastname: Arutyunov
authors.forename: Laura S
authors.initials: LS
authors.lastname: Frost
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
chemical_list: D001425:Bacterial Outer Membrane Proteins; C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; C041427:TraJ protein, bacteria; D049071:Endopeptidase Clp