Early events in the binding of the pPS10 replication protein RepA to single iteron and operator DNA sequences.
RepA protein, encoded in the Pseudomonas pPS10 replicon, is a stable dimer in solution (dRepA), acting as a self-repressor of repA transcription through binding to an inverted repeat operator. However, RepA monomers (mRepA) are required to initiate plasmid replication upon binding to four directly repeated DNA sequences (iterons). RepA is composed of two winged-helix (WH) domains: C-terminal WH2 is the main DNA-binding domain (DBD) for both target sequences, whereas N-terminal WH1 acts as dimerization interface in dRepA, but becomes a second DBD in mRepA. On the basis of CD spectroscopy, hydrodynamics, X-ray crystallography and model building studies, we proposed previously that the activation of RepA initiator implies a large structural change in WH1, coupled to protein monomerization and interdomain compaction. Here, we report novel features in the process. Binding curves of RepA to an iteron, followed by fluorescence anisotropy in solution and by surface plasmon resonance on immobilized DNA, exhibit the profiles characteristic of transitions between three states. In contrast, RepA-R93C, a monomeric activated mutant, exhibits a single binding transition. This suggests the presence of an intermediate species in the iteron-induced dissociation and structural transformation of RepA. High concentrations of bovine serum albumin or ovalbumin (macromolecular crowding) enhance RepA affinity for an iteron in solution and, in gel mobility-shift assays, result in the visualization of novel protein-DNA complexes. RepA-induced DNA bending requires the binding of two WH domains: either both WH2 in dimers (operator) or WH1 plus WH2 in monomers (iteron).
Díaz-López
Teresa
T
Department of Molecular Microbiology, Centro de Investigaciones Biológicas-CSIC, Madrid, Spain.
Dávila-Fajardo
Cristina
C
Blaesing
Franca
F
Lillo
M Pilar
MP
Giraldo
Rafael
R
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D008958:Models, Molecular; D009154:Mutation; D009875:Operator Regions, Genetic; D010047:Ovalbumin / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018741:Replication Origin; D012710:Serum Albumin, Bovine / Q000378:metabolism; D020349:Surface Plasmon Resonance; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D004247:DNA; D004265:DNA Helicases