Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision

Hinerfeld, D Churchward, G

D. Hinerfeld et G. Churchward, Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision, MOL MICROB, 41(6), 2001, pp. 1459-1467

42

INGLESE

Article

Microbiology

MOLECULAR MICROBIOLOGY

0950-382X → ACNP

41

6

2001

1459 - 1467

ISI

0950-382X(200109)41:6<1459:XPOTCT>2.0.ZU;2-2

The binding of Tn916Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis f ormed two complexes with different electrophoretic mobilities with both rig ht and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concen tration 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 i nterference was used to identify two G residues that were essential for bin ding 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 frequenc y of excision of a minitransposon from a plasmid, indicating that binding o f Xis at the right end of Tn916 inhibits transposon excision. A similar mut ation in the specific binding site for Xis at the left end of the transposo n did not reduce the affinity of Xis for the site but did perturb binding s ufficiently to alter the pattern of protection by Xis from nuclease cleavag e. 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 concentrat ions, can also regulate this process.