Subject(s)
Imidazoles/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Lymphoma, Follicular/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinolines/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Blotting, Western , Boronic Acids/pharmacology , Bortezomib , Cell Proliferation/drug effects , Female , Forkhead Transcription Factors/physiology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lymphoma, Follicular/metabolism , Lymphoma, Follicular/pathology , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazines/pharmacology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Sigma Factor/metabolism , Symporters , Trans-Activators/genetics , Transcriptional Activation , Aldose-Ketose Isomerases/genetics , Amino Acid Substitution , Amino Acids , Base Sequence , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Membrane Transport Proteins/genetics , Models, Molecular , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Conformation , Sigma Factor/genetics , Trans-Activators/chemistry , Trans-Activators/metabolismABSTRACT
RhaS, an AraC family protein, activates rhaBAD transcription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of rhaB-lacZ activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp -65 and -67 in rhaI1, and that Arg202 contacts -46 and Arg206 contacts -48 in rhaI2. In motif 2, we found that Asp250 and Asn252 both contact the bp -79 in rhaI1. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.
