Quaternary structural transitions in the DeoR-type repressor UlaR control transcriptional readout from the L-ascorbate utilization regulon in Escherichia coli.
UlaR is a DNA binding protein of the DeoR family of eubacterial transcriptional repressors which maintains the utilization of the L-ascorbate ula regulon in a repressed state. The availability of L-ascorbate in the growth medium releases UlaR-mediated repression on the ula regulon, thereby activating transcription. The molecular details of this induction by L-ascorbate have remained elusive to date. Here we have identified L-ascorbate 6-phosphate as a direct effector of UlaR; using a combination of site-directed mutagenesis, gel retardation, isothermal titration calorimetry, and analytical ultracentrifugation studies, we have identified the key amino acid residues that mediate L-ascorbate 6-phosphate binding and constructed the first model of regulation of a DeoR family member, establishing the basis of the ula regulon transcription control by UlaR. In this model, specific quaternary rearrangements of the DeoR-type repressor are the molecular underpinning of the activating and repressing forms. A DNA-bound UlaR tetramer establishes repression, whereas an L-ascorbate-6-phosphate-induced breakdown of the tetrameric configuration in favor of an UlaR dimeric state results in dissociation of UlaR from DNA and allows transcription of ulaG and ula ABCDEF structural genes. Despite the fact that similar changes have been described for other unrelated repressor factors, this is the first report to demonstrate that specific oligomerization changes are responsible for the activating and repressing forms of a DeoR-type eubacterial transcriptional repressor.
Garces
Fernando
F
Centro de Investigaciones Biológicas (CIB-CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Fernández
Francisco J
FJ
Gómez
Antonia M
AM
Pérez-Luque
Rosa
R
Campos
Evangelina
E
Prohens
Rafel
R
Aguilar
Juan
J
Baldomà
Laura
L
Coll
Miquel
M
Badía
Josefa
J
Vega
M Cristina
MC
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018085:Regulon; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C489070:DeoR protein, E coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012097:Repressor Proteins; D001205:Ascorbic Acid