Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the... Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli Jason R. Wickstrum 1 , † , Thomas J. Santangelo 2 , † ‡ , and Susan M. Egan 1 , * 1 Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045 2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850 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 (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Bacteriology American Society For Microbiology

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Journal of Bacteriology , Volume 187 (19): 6708 – Oct 1, 2005

Abstract

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli Jason R. Wickstrum 1 , † , Thomas J. Santangelo 2 , † ‡ , and Susan M. Egan 1 , * 1 Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045 2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850 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 (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-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.

Loading next page...
 
/lp/american-society-for-microbiology/cyclic-amp-receptor-protein-and-rhar-synergistically-activate-rXuFDkDunm

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
American Society For Microbiology
Copyright
Copyright © 2005 by the American society for Microbiology.
ISSN
0021-9193
eISSN
1098-5530
DOI
10.1128/JB.187.19.6708-6718.2005
pmid
16166533
Publisher site
See Article on Publisher Site

Abstract

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli Jason R. Wickstrum 1 , † , Thomas J. Santangelo 2 , † ‡ , and Susan M. Egan 1 , * 1 Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045 2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850 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 (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-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.

Journal

Journal of BacteriologyAmerican Society For Microbiology

Published: Oct 1, 2005

There are no references for this article.