Abstract
Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.
Highlights
Sleeping Beauty (SB) is the most active Tc1/marinerlike transposon in vertebrate species
We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the inverted repeats (IRs)/DR
We found that the two HTH subdomains and the GRPR-like motif are all contributing to DNA binding, with the N-terminal PAI subdomain and a 10-bp binding site core sequence having a dominant role in this process
Summary
Proach, we have reconstructed an active Tc1-like transposon from bits and pieces of inactive elements found in the genomes of teleost fish, and named this transposon Sleeping Beauty (SB) [12]. The transposase-binding sites of SB elements are repeated twice per IR in a direct orientation (DRs). This special organization of inverted repeat, termed IR/DR, is shared by a group of Tc1-like transposons, but not by Tc1 itself [13]. We carried out a functional analysis of the DNA-binding domain of the transposase and DNA sequence motifs within the inverted repeats of SB transposons. We found that the two HTH subdomains and the GRPR-like motif are all contributing to DNA binding, with the N-terminal PAI subdomain and a 10-bp binding site core sequence having a dominant role in this process. The PAI subdomain binds to a transpositional enhancer-like sequence within the left inverted repeat of SB and mediates protein-protein interactions with other transposase subunits. Enhanced stability of synaptic complexes is one possible explanation for the efficient transposition of Sleeping Beauty in vertebrate cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
