The protein–DNA contacts in RutR·carAB operator complexes

Abstract

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix–turn–helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.

A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

“…Alternatively, contact between MdrA and RNAP may be mediated by additional elements that could introduce conformational changes in chromatin structure. Similar mechanisms were previously suggested for other positive regulators that bind far upstream from the transcription start site but whose mechanisms of activation have not yet been elucidated (e.g., EspR, RutR, TodT, and AlgR) (20,26,29,39). It is worth mentioning that the possible involvement of elements that introduce modifications of the nucleoprotein structure of local sequences was also hypothesized for the LuxR-type regulator VjbR, since it binds at position Ϫ94 and consequently could not activate transcription of the virB promoter in a prototypical manner (2).…”

Ligand binding specificity of RutR, a member of the TetR family of transcription regulators inEscherichia coli

“…Previously we have shown that high affinity binding of RutR to the car P1 control region is inhibited in the presence of uracil, whereas thymine has little or no effect [12] . This contrasts with an earlier observation [9] and suggests that thymine might be selectively excluded from the effector-binding site.…”

The TetR Family of Regulators

“…A homolog of RutR, PydR, has been identified as the regulator of genes required for pyrimidine degradation via the reductive pathway in Pseudomonas putida (154). A crystal structure is available for RutR in complex with its inducing ligand uracil (155). Residues involved in uracil binding are conserved in PydR, indicating that PydR may also be a uracil-responsive transcription factor (154).…”