TY - JOUR
T1 - Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor
AU - O'Connor, Tamara J.
AU - Nodwell, Justin R.
N1 - Funding Information:
We thank Linda Kenney for supplying us with purified OmpR and phosphoramidate and for her helpful advice. We also thank Nancy Sheeler for supplying us with purified AbsA2, Kalinka Koteva for expert technical assistance with the mass spectrometric analysis of RamR, Denis Daigle for helpful advice and expert technical assistance, Eric Brown, Gerry Wright and Yingfu Li for use of laboratory equipment and software. We also thank Mark Buttner, Denis Daigle and Andrew Willems for careful review of this manuscript. This work was funded by a New Investigator Award and an Operating Grant no. MOP-68817 from the Canadian Institute of Health Research to J.N. and an Ontario Graduate Scholarship to T.O.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2005/9/2
Y1 - 2005/9/2
N2 - The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.
AB - The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.
KW - Development
KW - Phosphorylation
KW - RamR
KW - Response regulator
KW - Streptomyces
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U2 - 10.1016/j.jmb.2005.06.053
DO - 10.1016/j.jmb.2005.06.053
M3 - Article
C2 - 16051268
AN - SCOPUS:23644457186
SN - 0022-2836
VL - 351
SP - 1030
EP - 1047
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 5
ER -