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

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Abstract
RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response tol-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether ς70 might be an activation target for RhaS. We found that ς70 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 ς70residues 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 ς70 substitutions and found that RhaS D241 and ς70 R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that ς70 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 ς70. More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with ς70 may be conserved.