The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control.
The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.
Matsunaga
F
F
Institute for Virus Research, Kyoto University, Japan.
Ishiai
M
M
Kobayashi
G
G
Uga
H
H
Yura
T
T
Wada
C
C
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D019281:Dimerization; D029968:Escherichia coli Proteins; D005144:F Factor / Q000187:drug effects / Q000502:physiology*; D018628:Gene Dosage; D018832:Molecular Chaperones / Q000502:physiology; D016254:Mutagenesis, Insertional; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C074164:RepE protein, E coli; D012097:Repressor Proteins