Changing the recognition site of a conjugative relaxase by rational design |
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Authors: | Blanca González-Pérez José Daniel Carballeira Gabriel Moncalián Fernando de la Cruz Professor |
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Institution: | Departamento de Biología Molecular, Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-IDICAN), Santander, Spain |
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Abstract: | TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA–TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their conjugation efficiency. |
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Keywords: | Bacterial conjugation Rational design Relaxase |
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