(A)Model of R2 TPRT. If element RNA is not present in the reaction, the purified protein exposes both DNA binding domains, N-terminal (blue line) and C-terminal (red line) enabling the protein to contact two DNA molecules at a time, generating an interlaced matrix of complexes that becomes stuck in the well on EMSA gels. Protein bound to the 3’ UTR RNA sequesters the N-terminal domain, exposing only the C-terminal DNA binding domain (i.e., the “upstream subunit” conformation). Protein bound to the 5’ RNA sequesters the C-terminal DNA binding domain, exposing only the N-terminal DNA binding domain to adopt the “downstream subunit” conformation.

(B)The R2 protein uses a dimer bound to target DNA in opposite orientations to affect transposition. The upstream subunit binds to DNA sequences upstream of the insertion site while the downstream subunit binds to sequences downstream of the insertion site. A complete TPRT reaction occurs in four discrete steps;  (Step 1) the endonuclease (red oval) from the first upstream is responsible for generating the nick that will be used to prime first strand TPRT.  (Step 2) The RT (green oval) of the upstream subunit catalyzes first strand TPRT. (Step 3) The downstream subunit supplies the endonuclease (red oval) used to cleave the remaining DNA strand. Second strand cleavage requires the R2 subunit be void of element RNA, forcing cleavage to occur after first strand TPRT . (Step 4) The downstream subunit is thought to provide the polymerase (green oval) used to perform second strand TPRT, although no evidence of this step has been observed in vitro.