Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA.
Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.
Ravin
Nikolai V
NV
Bioengineering Centre, Russian Academy of Sciences, 7-1 Prosp. 60 let Oktiabria, 117312 Moscow, Russian Federation.
Rech
Jérôme
J
Lane
David
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins; D005144:F Factor / Q000235:genetics* / Q000502:physiology; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D009876:Operon; D010641:Phenotype; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C060972:SopB protein, Bacteria; C489223:SopB protein, E coli; C060971:SopA protein, Bacteria