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Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and
1Institute for Virus Research, Kyoto University, Japan.
Abstract:
Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.
Insights
RepE protein monomers initiate mini-F plasmid replication by binding ori2, while RepE dimers repress transcription by binding the operator. Monomerization activates RepE
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Replication of mini-F plasmid depends on RepE initiator protein and Escherichia coli heat shock proteins (DnaJ, DnaK, GrpE).
- RepE protein has dual functions: initiating replication at ori2 and repressing its own transcription.
- A mini-F plasmid mutant yields altered RepE (RepE54) with enhanced initiation but reduced repressor activity, capable of replicating in dnaJ-defective hosts.
Purpose of the Study:
- To investigate the functional differentiation of the RepE protein in mini-F plasmid replication and regulation.
- To determine the role of RepE monomeric and dimeric forms in DNA binding and replication initiation/repression.
Main Methods:
- Purification of wild-type RepE and mutant RepE54.
- Gel-retardation assays to assess binding affinities to ori2 and repE operator.
- Treatment of wild-type RepE with denaturants to induce monomerization.
Main Results:
- RepE54 was purified mainly as monomers, showing high affinity for ori2 but low affinity for the repE operator.
- Wild-type RepE dimers, upon denaturation, partially converted to monomers, enhancing ori2 binding and reducing operator binding.
- ori2 binding activity was exclusively associated with RepE monomers.
Conclusions:
- RepE monomers are the active form for initiating mini-F plasmid replication via ori2 binding.
- RepE dimers function as repressors by binding to the repE operator.
- RepE monomerization, potentially mediated by heat shock proteins, activates initiator function and regulates mini-F DNA replication.