Journal Article

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

Sven Sewitz, Paul Crellin and Ronald Chalmers

in Nucleic Acids Research

Volume 31, issue 20, pages 5868-5876
Published in print October 2003 | ISSN: 0305-1048
Published online October 2003 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkg797
The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

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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 α and β sides of the complex. IHF can dissociate from the transposon arm on the β side of the complex in the absence of metal ion. However, IHF is locked onto the α 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.

Journal Article.  7165 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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