Changing the recognition site of a conjugative relaxase by rational design.
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.
González-Pérez
Blanca
B
Departamento de Biología Molecular, Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria, Santander, Spain.
Carballeira
José Daniel
JD
Moncalián
Gabriel
G
de la Cruz
Fernando
F
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003227:Conjugation, Genetic; D003545:Cysteine / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity / Q000235:genetics; D014633:Valine / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase; C510362:TrwC protein, E coli; D014633:Valine; D003545:Cysteine; D000409:Alanine