The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.
The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.
Wikström
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P
Institute for Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
O'Neill
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E
Ng
L C
LC
Shingler
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V
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002918:Chymotrypsin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol; D006868:Hydrolysis / Q000187:drug effects; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000737:chemistry* / Q000201:enzymology; D012492:Salts / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C080133:DmpR protein, Pseudomonas; D012492:Salts; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D004247:DNA; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; D005990:Glycerol