title: Mechanism of ToxT-dependent transcriptional activation at the Vibrio cholerae tcpA promoter.
abstract: The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae. ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others. In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from -95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to -63. A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation. Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer. Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit. A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at -72 and -51 and requires the alpha C-terminal domain of RNA polymerase.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hulbert
authors.forename: Ronald K
authors.initials: RK
authors.lastname: Taylor
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002771:Cholera / Q000382:microbiology; D002772:Cholera Toxin; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D036781:Fimbriae Proteins; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C085807:TcpA protein, Vibrio cholerae; D014157:Transcription Factors; D036781:Fimbriae Proteins; C075626:tcpN protein, Vibrio cholerae; D002772:Cholera Toxin; D012321:DNA-Directed RNA Polymerases