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Journal of Bacteriology, September 2000, p. 4959-4969, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genetic Evidence that Transcription Activation by RhaS Involves Specific Amino Acid Contacts with Sigma 70

Prasanna M. Bhende and Susan M. Egan*

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received 21 April 2000/Accepted 9 June 2000

RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether sigma 70 might be an activation target for RhaS. We found that sigma 70 K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged sigma 70 residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and sigma 70 substitutions and found that RhaS D241 and sigma 70 R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that sigma 70 R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact sigma 70. More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with sigma 70 may be conserved.

* Corresponding author. Mailing address: 8031 Haworth Hall, Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045. Phone: (785) 864-4294. Fax: (785) 864-5294. E-mail: sme{at}ukans.edu.

Journal of Bacteriology, September 2000, p. 4959-4969, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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