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Inhibition of IS2 transposition by factor for inversion stimulation

Lei, Guang-Sheng, Chen, Chii-Jaan, Yuan, Hanna S., Wang, Shao-Hung, Hu, Shiau-Ting
FEMS microbiology letters 2007 v.275 no.1 pp. 98-105
DNA, Escherichia coli, binding sites, deoxyribonuclease I, gels, genes, luciferase, mutants, promoter regions, terminal repeat sequences, transcription (genetics), transposition (genetics)
The effect of factor for inversion stimulation (Fis) protein on IS2 transposition was investigated. A full-length IS2 was found to transpose at a frequency 64 times lower in a normal Escherichia coli than in a fis⁻ mutant. To investigate whether Fis affects IS2 transposition by DNA binding, gel retardation and DNase I footprinting experiments were performed. Analysis of Fis binding to the left terminus of IS2 revealed that Fis binds to nucleotide number 44-60 located between the -35 and -10 regions of the major IS2 promoter. To further determine whether Fis binding affects IS2 transcription, the major IS2 promoter was fused to a luciferase gene and assayed for its transcription efficiency in the presence or absence of Fis. The results showed that Fis reduced transcription from the major IS2 promoter by approximately sixfold. Analysis of Fis binding to the right terminal repeat of IS2 revealed that Fis binds to the inner end of the repeat, which is the same region as the place where the IS2 transposase binds. These results suggest that Fis inhibits IS2 transposition by blocking the binding sites of IS2 transposase and by repressing the transcription of IS2 genes.