title: Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision.
abstract: The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.
authors.affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hinerfeld
authors.forename: G
authors.initials: G
authors.lastname: Churchward
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003227:Conjugation, Genetic; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D013204:Staphylococcus Phages / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins
chemical_list: D004251:DNA Transposable Elements; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; C063911:excisionase