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PMID:16166533

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Citation

Wickstrum, JR, Santangelo, TJ and Egan, SM (2005) Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L-rhamnose-responsive rhaSR promoter in Escherichia coli. J. Bacteriol. 187:6708-18

Abstract

The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position -111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the alpha-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

Links

PubMed PMC1251584 Online version:10.1128/JB.187.19.6708-6718.2005

Keywords

Base Sequence; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; DNA-Directed RNA Polymerases/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutation; Promoter Regions, Genetic/genetics; Receptors, Cyclic AMP/metabolism; Rhamnose/metabolism; Trans-Activators/genetics; Trans-Activators/metabolism; Transcription, Genetic/physiology

Significance

Annotations

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

ECOLI:RHAR

acts_upstream_of_or_within

GO:0006351: transcription, DNA-templated

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:RHAR

enables

GO:0043565: sequence-specific DNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:CRP

acts_upstream_of_or_within

GO:0006351: transcription, DNA-templated

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:CRP

involved_in

GO:0006351: transcription, DNA-templated

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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