Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces.
The pi35.0 protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the gamma origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the pi protein and their heterodimers revealed that the predominant species, pi35.0 (35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while pi30.5 (30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a pi35.0 dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by pi35.0 and DNA fragments containing all seven DRs revealed coupled ("hand-cuffed") DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of pi35.0 in such structures. The pi protein (pi35.0, pi30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S (pir200), introduced into pi30.5 severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of pi35.0 dimers but it does not change the ability of pi35.0 to bind IRs. To explain these observations we propose that the diverse interactions of pi variants with DNA are controlled by multiple surfaces for protein oligomerization.
Urh
M
M
Department of Bacteriology, University of Wisconsis, Madison, WI 53706, USA.
Wu
J
J
Wu
J
J
Forest
K
K
Inman
R B
RB
Filutowicz
M
M
D000595:Amino Acid Sequence; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases