The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB-TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB-TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.photocrosslinking | protein-protein interaction | transpososome | transposable element

Tn7 is a transposable element isolated from E. coli that encodes five transposon‐specific proteins, Tns A, B, C, D, and E. TnsC is essential in the formation and activity of a Tn7 nucleoprotein complex comprised of TnsA,B,C, and D proteins with donor DNA as well as target DNA. The goal of this work is to identify and characterize the functional domains of TnsC. The Tn7 reaction mechanism suggests an interaction between TnsB and C. Crosslinking experiments were performed with a TnsB peptide and TnsC to stabilize and capture the complex. The data show that TnsB and TnsC participate in a direct physical interaction. Mass spectrometry of the complex will show which residues of TnsC interact with the peptide, and thereby reveal the TnsBC domain. In order to determine the remaining functional domains, Tn7 was used as a mutagen to generate a TnsC mutant library. The mutants were screened for transposition activity in a promoter capture assay. The mutable regions of TnsC were mapped and dominant negative mutants that, presumably, retain one or more essential activities of TnsC were identified. Future experiments will reveal which activity and, correspondingly, functional domain is disrupted. Experiments that identify the functional domains of TnsC will provide deeper insights into how Tn7 and other nucleoprotein complexes are controlled. This work was supported by Howard Hughes Medical Institute.