REPA1, A REPLICATION CONTROL PROTEIN OF THE REPFIC REPLICON OF PLASMID ENTP307. Simon E. V. Phillips, Haiwei Song, Renata Maas^*, Mark R. Parsons, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK, ^*Department of Microbiology, New York University Medical Center, New York, New York 10016, USA

The large E. Coli plasmid EntP307 contains two replication regions or basic replicons, RepFIB and RepFIC. Replication control in RepFIC is similar to that in plasmids R1 and R100, and is effected by only two structural genes. These encode the negative regulator RepA2, and the replication initiator RepAl. In addition, constitutive transcription from another promoter produces antisense RNA that negatively regulates RepA1 production. RepA1 probably interacts directly with the replication origin, but this has been difficult to demonstrate.

We have crystallized RepA1 to establish a structural basis for its function, and provide a prototype for Rep proteins of this class. It was purified using a modification of the published procedure¹, and crystallized by hanging drop vapour diffusion. Type 1 crystals² grow from 2.0M ammonium sulphate solutions at pH 8.5, and are orthorhombic P2₁2₁2, with a=61, b=67 and c=243Å. Monoclinic Type 2 crystals grow from PEG8000 solutions at lower pH, with a=64, b=103, c=65Å and [[beta]]=97deg.. Both forms contain two 40kDa RepA1 molecules per asymmetric unit, and diffract X-rays to about 3Å resolution. Preliminary data have been collected from native type 1 crystals at 100K, and from a crystal soaked in PCMBS which shows two major mercury sites. High resolution data collection is under way using synchrotron radiation.

1. Maas, R. et al (1991) Mol. Microbiol. 5, 927-932.

2. Song et al (1996) Proteins: Struct. Funct. Genet. in press.