Tn10 Transposase Mutants with Altered Transpososome Unfolding Properties are Defective in Hairpin Formation

Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction

“…To display the protection pattern, a molecular model of IHF-folded arm of Tn 10 was developed by combining the structure of the related Tn 5 transposase dimer with that of the IHF co-crystal (Figure 1C). The contacts in the model fit well with the hydroxyl radical protection pattern and together with several other lines of evidence support the model as an accurate representation of the general shape of the Tn 10 transpososome (25–27,32). The molecular model also illustrates the spatial relationship between the terminal and subterminal transposase contacts that define the IHF-loop.…”

“…However, very little may be known about the precise function or molecular mechanism of the various components, or the conformational changes that may take place at different stages of the reaction. Fortunately, several unique characteristics of the Tn 10 transposition system have allowed us to address the nature and function of conformational changes at different stages of the reaction using traditional molecular biological techniques (24–27). Key to this progress has been the insight provided by the crystal structure of the related Tn 5 transpososome (28).…”

“…Although the precise significance of this is unknown, it almost certainly reflects a conformational change in preparation for the first chemical step of the reaction, a nick located between the two hypersensitive nucleotides. Further evidence that unfolding of the IHF-loop promotes conformational changes is provided by the point mutations RA182 and RA184, which both have altered unfolding properties and block the formation of the hairpin intermediate (27). However, the most direct evidence is that truncation of the α transposon arm, to deprive the complex of the most distal set of subterminal contacts, blocks hairpin resolution and stalls the reaction at the SEB stage (26).…”

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

“…It is also notable that H-NS did not have a significant effect on excision, because previous work has provided evidence that full excision is coupled to transpososome unfolding (Crellin et al 2004;Humayun et al 2005). Our failure in the present study to observe an H-NS effect on transposon excision could either be an indication that transpososome unfolding is a limiting step only at the transition from fully cleaved transpososome to strand transfer, or that full excision and transpososome unfolding are not actually coupled.…”

The Nucleoid Binding Protein H-NS Acts as an Anti-Channeling Factor to Favor Intermolecular Tn10 Transposition and Dissemination

“…9 H-NS may promote OE unfolding by either competing with IHF for the IHF binding site or binding close to this site and altering either the conformation of the site or the trajectory of the IHF bend such that the potential of the ST region of the OE to contact transposase is reduced. OE folding and unfolding have also been implicated in steps in Tn10 excision; 19,20 however, H-NS does not impact on excision in the linear fragment system. 9 We have not previously examined the impact of H-NS on transposition reactions performed with supercoiled Tn10 substrate plasmids in vitro.…”