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A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain

Nature Structural & Molecular Biology volume 10, pages 565–571 (2003)Cite this article

Abstract

Plasmids are natural vectors for gene transfer. In Gram-negative bacteria, plasmid DNA replication is triggered when monomers of an initiator protein (Rep) bind to direct repeats at the origin sequence. Rep dimers, which are inactive as initiators, bind to an inverse repeat operator, repressing transcription of the rep gene. Rep proteins are composed of N-terminal dimerization and C-terminal DNA-binding domains. Activation of Rep is coupled to dimer dissociation, converting the dimerization domain into a second origin-binding module. Although the structure of the monomeric F plasmid initiator (mRepE) has been determined, the molecular nature of Rep activation remains unknown. Here we report the crystal structure of the dimeric N-terminal domain of the pPS10 plasmid initiator (dRepA). dRepA has a winged-helix fold, as does its homologous domain in mRepE. However, dimerization transforms an interdomain loop and β-strand (monomeric RepE) into an α-helix (dimeric RepA). dRepA resemble the C terminus of eukaryotic and archaeal Cdc6, giving clues to the phylogeny of DNA replication initiators.

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Figure 1: Overall architecture of dRepA.
Figure 2: Stereo view of the details of dRepA structure.
Figure 3: Comparison of dRepA and mRepE reveals structural differences linked to Rep activation.
Figure 4: A schematic diagram of the conformational activation of Rep proteins.
Figure 5: Eukaryotic/archaeal replication initiator mCdc6 is structurally related to bacterial dRepA.

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Acknowledgements

We thank the staff at beamline X31 in EMBL-DESY for help with data collection. We are indebted to D. Rhodes for the critical reading of the manuscript and, together with V. Ramakrishnan, for encouragement. This work has been financed with grants of Spanish MCyT, CAM and a FIS network for research on infectious pathology.

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Authors and Affiliations

  1. Department of Molecular Microbiology Centro de Investigaciones Biológicas–CSIC, C/ Velázquez 144, Madrid, 28006, Spain

    Rafael Giraldo & Ramón Díaz-Orejas

  2. Department of Protein Structure and Function Centro de Investigaciones Biológicas–CSIC, Centro de Investigaciones Biológicas–CSIC, C/ Velázquez 144, Madrid, 28006, Spain

    Carlos Fernández-Tornero & Antonio Romero

  3. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Philip R Evans

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Correspondence to Rafael Giraldo.

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Giraldo, R., Fernández-Tornero, C., Evans, P. et al. A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain. Nat Struct Mol Biol 10, 565–571 (2003). https://doi.org/10.1038/nsb937

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