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PMID:11849541

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Citation

Yu, RR and DiRita, VJ (2002) Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol. Microbiol. 43:119-34

Abstract

Co-ordinate expression of many virulence genes in the human pathogen Vibrio cholerae is under the direct control of the ToxT protein, including genes whose products are required for the biogenesis of the toxin-co-regulated pilus (TCP) and cholera toxin (CTX). This work examined interactions between ToxT and the promoters of ctx and tcpA genes. We found that a minimum of three direct repeats of the sequence TTTTGAT is required for ToxT-dependent activation of the ctx promoter, and that the region from -85 to -41 of the tcpA promoter contains elements that are responsive to ToxT-dependent activation. The role of H-NS in transcription of ctx and tcpA was also analysed. The level of activation of ctx-lacZ in an E. coli hns- strain was greatly increased even in the absence of ToxT, and was further enhanced in the presence of ToxT. In contrast, H-NS plays a lesser role in the regulation of the tcpA promoter. Electrophoretic mobility shift assays demonstrated that 6x His-tagged ToxT directly, and specifically, interacts with both the ctx and tcpA promoters. DNase I footprinting analysis suggests that there may be two ToxT binding sites with different affinity in the ctx promoter and that ToxT binds to -84 to -41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT-dependent gene expression, ToxT binds to AT-rich promoters that may have a specific secondary conformation, displaces H-NS and stimulates RNA polymerase resulting in transcription activation.

Links

PubMed

Keywords

Bacterial Outer Membrane Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Binding Sites; Cholera Toxin/genetics; DNA Fingerprinting/methods; DNA, Bacterial; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; DNA-Directed RNA Polymerases/metabolism; Deoxyribonuclease I; Escherichia coli/genetics; Fimbriae Proteins; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Genes, Bacterial; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Repressor Proteins/genetics; Repressor Proteins/metabolism; Transcription Factors/metabolism; Transcriptional Activation; Vibrio cholerae/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

VIBC3:TCPN

enables

GO:0000976: transcription regulatory region sequence-specific DNA binding

ECO:0001807: electrophoretic mobility shift assay evidence used in manual assertion

F

Seeded From UniProt

complete

VIBC3:TCPN

enables

GO:0000976: transcription regulatory region sequence-specific DNA binding

ECO:0005631: DNAse footprinting evidence used in manual assertion

RefSeq:NC_009456.1

F

Seeded From UniProt

complete

VIBC3:TCPN

enables

GO:0001216: DNA-binding transcription activator activity

ECO:0001807: electrophoretic mobility shift assay evidence used in manual assertion

F

Seeded From UniProt

complete

VIBC3:TCPN

enables

GO:0001216: DNA-binding transcription activator activity

ECO:0005631: DNAse footprinting evidence used in manual assertion

RefSeq:NC_009456.1

F

Seeded From UniProt

complete

VIBC3:TCPN

part_of

GO:0032993: protein-DNA complex

ECO:0001807: electrophoretic mobility shift assay evidence used in manual assertion

C

Seeded From UniProt

complete

VIBC3:TCPN

part_of

GO:0032993: protein-DNA complex

ECO:0005631: DNAse footprinting evidence used in manual assertion

RefSeq:NC_009456.1

C

Seeded From UniProt

complete

VIBC3:TCPN

involved_in

GO:0045893: positive regulation of transcription, DNA-templated

ECO:0001204: in vitro transcription assay evidence used in manual assertion

P

Seeded From UniProt

complete

Notes

See also

References

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