IHF Modulation of Tn10 Transposition: Sensory Transduction of Supercoiling Status via a Proposed Protein/DNA Molecular Spring

Abstract

Architectural protein IHF modulates Tn10 transposition in vitro. IHF stimulates transposon excision. Also, separately, IHF forces transposon end/target DNA interactions into a constrained pathway, "channeling," that yields only unknotted intratransposon inversion circles. Negative supercoiling influences both effects, differently. We infer that IHF is an architectural catalyst: it promotes initial transpososome assembly and is then ejected from the transpososome. IHF then rebinds, altering transpososome conformation to promote channeling. We also infer that the developing transpososome is a molecular spring: DNA provides basic elasticity; a conformational change in transposase provides force; and IHF and/or supercoiling provide conformational inputs. In vivo, IHF is a sensory transducer of chromosomal supercoiling status: with supercoiling absent, IHF is "supercoiling relief factor"; with supercoiling present, stimulation and channeling comprise a homeostatic pair such that modest changes in chromosome condition strongly influence transpositional outcome.

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

“…hns12 clones 1-3 and clone 1 from the wild-type strain). The qualitative and quantitative changes in the STP species are indicative of a transpososome structure in which the transposon ends are less tightly constrained relative to the folded transpososome (Chalmers et al 1998). Furthermore, since these changes are dependent on a mutation in H-NS, the results support the idea that H-NS plays an important role in modulating the structure of the transpososome in vivo.…”

“…The latter products are almost exclusively unknotted inversion circles (Benjamin and Kleckner 1989). These intratransposon events arise as a consequence of geometric and topological constraints imposed by DNA supercoiling and IHF binding to the outside ends of the transpososome (Chalmers et al 1998). If H-NS does influence the structure of the transpososome in vivo, as we have provided evidence for in vitro, then some mutant forms of H-NS might produce a different distribution of STPs.…”

“…1B; Chalmers et al 1998). It follows that the types of STPs observed provide information regarding the conformation of the fully cleaved transpososome.…”

“…In contrast, when IHF is not bound to the cleaved transpososome, the above constraint is not imposed and intermolecular insertion events are favored. Thus, in addition to promoting Tn10 transposition by facilitating transpososome assembly and full excision, IHF can also inhibit intermolecular transposition events (Chalmers et al 1998).…”

Questions and Assays

“…Physical and biochemical studies confirmed this conclusion and revealed substeps and mechanistic details (e.g., Bolland and Kleckner 1996;Sakai and Kleckner 1997), including work with the Haniford group identifying a hairpin intermediate, which linked the mechanism of transposition to that of immunoglobulin rearrangements (Kennedy et al 1998). We also showed that Tn10 transposition is regulated by the imposition and release of torsional mechanical stress within a small DNA loop at one end of the transposon, with interplay between a bend-inducing architectural protein (IHF) and cellular supercoiling (Chalmers et al 1998). With this study, we first put our toes into the water of chromosomes as mechanical objects.…”