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The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control1

https://doi.org/10.1006/jmbi.1997.1373Get rights and content

Abstract

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.

Introduction

The mini-F plasmid, a derivative of the Escherichia coli F-factor, is maintained at one to two copies per host chromosome. Its replication initiation is provoked by binding of the plasmid-coded RepE protein (251 residues, 29 kDa) to the origin, ori2 Masson and Ray 1986, Tokino et al 1986, Muraiso et al 1987, Kawasaki et al 1996. RepE also plays a regulatory role in replication by assigning two distinctive functions to the monomer and the dimer: the monomer binds to each of four directly repeated iterons (DR, direct repeat) of ori2 to initiate replication, whereas the dimer binds to the inverted repeat, the operator of the repE gene, to autogenously repress its transcription catalyzed by RNA polymerase containing σ32(Wada et al 1987, Masson and Ray 1988, Ishiai et al 1994; see Figure 1).

As expected from the previous observations that RepE exists mostly as dimers (Masson & Ray, 1988), purified RepE forms stable dimers (dissociation constant 0.3 nM; Ishiai et al., 1994). The requirement of molecular chaperones (DnaK, DnaJ and GrpE; these proteins function synergistically) for mini-F replication Ezaki et al 1989, Kawasaki et al 1990 may thus be explained by assuming that they facilitate conversion of RepE dimers to monomers. In analogous studies with mini-P1 plasmid, the RepA initiator protein (dimer) was shown to be converted by the same set of chaperones to monomers and become active in replication (for reviews, see Baker and Wickner 1992, Chattoraj 1995). However, the functional structure of the initiator proteins of mini-F and related plasmids has not been studied in detail. Specifically, the structural basis for differentiating the dual functions of RepE must be examined to further understand the regulatory mechanism of mini-F replication.

In spite of the structural differences between the direct repeat and the inverted repeat, they share a common 8 bp sequence which may determine the binding specificity for RepE (Figure 1). The C-terminal region (residues 168 to 242) of RepE was recently shown to be primarily responsible for binding to both the origin and the operator, most probably by forming a DNA-binding domain(see Figure 7, below; Matsunaga et al., 1995). Furthermore, the region spanning residues 93 to 135 was shown to be involved in the negative regulation of replication, possibly by facilitating dimerization, because the chaperone-independent copy-up mutations were isolated specifically from this region Kawasaki et al 1991, Ishiai et al 1992. One such mutant protein, RepE54 (Arg118 changed to Pro; R118P) was found as stable monomers (Ishiai et al., 1994), suggesting that this region is important for dimerization. On the other hand, the N-terminal region of RepE (residues 21 to 55) has a conserved leucine-zipper-like sequence (Giraldo et al., 1989), a typical dimerization motif which is involved in dimerization at least in plasmid pPS10 (Garcı́a de Viedma et al., 1996). In the R6K plasmid, the N-terminal half of the initiator protein π can form dimers (Levchenko et al., 1994), and this region contains a leucine-zipper-like motif and another motif (RGD) which may also participate in protein-protein interaction (York & Filutowicz, 1993).

Here, we attempted to determine the region(s) of RepE involved in dimerization, and in particular those that may serve as an interface in dimerization by the isolation of RepE mutants defective in repressor activities and their characterization.

Section snippets

Isolation of RepE mutants defective in autogenous repression

Because RepE dimers, but not monomers, act as repressor of repE transcription, and a class of mutants affected in repression are expected to be deficient in dimerization, we screened for RepE mutants defective in repression. The repressor activity of mutant RepE was monitored by using derivatives of strain KY1461 or MC4100 carrying λ prophage which contained a PrepE-lacZ reporter gene, a fusion between the promoter/operator of repE and lacZ (Kawasaki et al., 1990). These lysogens (KY1463 and

Bacterial strains, phage and plasmids

Bacterial strains, phage and plasmids used are listed in Table 1. KY1463 and KY1464 were a λPrepE-lacZ lysogen of KY1461 and MC4100, respectively. KY1465 was constructed by introducing ΔdnaK53∷CmR from JC7623 carrying the dnaK null mutation by transduction with phage P1vir. The construction of λPrepE-lacZ was reported (Kawasaki et al., 1991). The expression of RepE from plasmid pKV7190, pKV7203 and pBK815 was regulated by the trp promoter, a modified phage T5 promoter, and a phage T7 promoter,

Acknowledgements

We are grateful to T. Nagata for critical reading of the manuscript and M. Mihara for laboratory supplies. We thank B. C. Kline for his gift of plasmids pBK815, pBK819 and pBK820. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan.

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    Edited by M. Gottesman

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    Present addresses: M. Ishiai, Institute for Hepatic Research, Kansai Medical University, Osaka 570 Japan

    3

    T.Yura, HSP Research Institute, Kyoto Research Park, Kyoto 606, Japan.

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