The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms.
The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.
Sewitz
Sven
S
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Crellin
Paul
P
Chalmers
Ronald
R
D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods; D004251:DNA Transposable Elements; D024202:Electrophoretic Mobility Shift Assay; D017665:Hydroxyl Radical / Q000737:chemistry; D034982:Integration Host Factors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D019895:Transposases / Q000235:genetics
D002413:Cations, Divalent; D004251:DNA Transposable Elements; D034982:Integration Host Factors; D017665:Hydroxyl Radical; D004247:DNA; D019895:Transposases