Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

摘要: Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential transfer. bind cleave one strand site- sequence-specifically before of the cleaved strand. TraI36, a domain F TraI that contains relaxase activity, binds sequence in single-stranded form with subnanomolar KD high specificity. Despite 91% amino acid identity, TraI36 domains from plasmids R100 discriminate between binding sites. The sites differ by 2 11 bases, but both their cognate site three orders magnitude higher affinity than other site. To identify specificity determinants, we generated variants having acids background. Although most retain specificity, Q193R/R201Q variant 10-fold greater reverse switch (R193Q/Q201R) confers wild-type on variant. Nonadditivity individual base contributions to recognition suggests difference derives multiple interactions. crystal structure shows positions 193 201 opposite sides pocket within cleft, suggesting involves knob-into-hole Specificity presumably modulated altering composition pocket. Our results demonstrate F-like relaxases can highly sequence-specific different sequences minimal substitution.