Fluorescence studies of the replication initiator protein RepA in complex with operator and iteron sequences and free in solution.
RepA, the replication initiator protein from the Pseudomonas plasmid pPS10, regulates plasmid replication and copy number. It is capable of autorepression, in which case it binds as a dimer to the inverted repeat operator sequence preceding its own gene. RepA initiates plasmid replication by binding as a monomer to a series of four adjacent iterons, which contain the same half-repeat as found in the operator sequence. RepA contains two domains, one of which binds specifically to the half-repeat. The other is the dimerization domain, which is involved in protein-protein interactions in the dimeric RepA-operon complex, but which actually binds DNA in the monomeric RepA-iteron complex. Here, detailed fluorescence studies on RepA and an N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-labeled single-cysteine mutant of RepA (Cys160) are described. Using time-resolved fluorescence depolarization measurements, the global rotational correlation times of RepA free in solution and bound to the operator and to two distinct iteron dsDNA oligonucleotides were determined. These provide indications that, in addition to the monomeric RepA-iteron complex, a stable dimeric RepA-iteron complex can also exist. Further, Förster resonance energy transfer between Trp94, located in the dimerization domain, and N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-Cys160, located on the DNA-binding domain, is observed and used to estimate the distance between the two fluorophores. This distance may serve as an indicator of the orientation between both domains in the unbound protein and RepA bound to the various cognate DNA sequences. No major change in distance is observed and this is taken as evidence for little to no re-orientation of both domains upon complex formation.
Diederix
Rutger E M
RE
Departamento de Biofísica, Instituto de Química Física Rocasolano, CSIC, Madrid, Spain.
Dávila
Cristina
C
Giraldo
Rafael
R
Lillo
M Pilar
MP
D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D009841:Oligonucleotides / Q000378:metabolism*; D009875:Operator Regions, Genetic; D010957:Plasmids; D011485:Protein Binding; D012399:Rotation; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D012996:Solutions; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases