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Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR

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

Received 17 May 2004/ Accepted 24 June 2004

The RhaS and RhaR proteins are transcription activators that respond to the availability of L-rhamnose and activate transcription of the operons in the Escherichia coli L-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the –35 hexamers of the promoters they regulate, suggesting they may contact the ⁷⁰ subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and ⁷⁰ residue R599. In the present study, we first identified two positively charged amino acids in ⁷⁰, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and ⁷⁰ R599. Finally, previous results from our lab separately showed that RhaS D250A and ⁷⁰ K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and ⁷⁰.

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