Chapter 18 : Tn5 Transposition

Tn5structure. (A) Tn5. Two nearly identical insertion sequences (IS50L and IS50R) bracket three genes that encode resistance to kanamycin, bleomycin, and streptomycin. IS50R encodes two proteins that play important roles in Tn5transposition: the transposase (Tnp) and the inhibitor of transposition (Inh). Both proteins are read in the same reading frame, but Inh is initiated 56 residues downstream of Tnp. IS50L encodes two inactive truncated versions of these proteins (P3 and P4) because of an ochre codon 26 residues from the C terminus. The cissites that define the ends of the transposable elements and at which Tnp acts are 19-bp sequences called OE (outside end) and IE (inside end) ( Fig. 1D ). For Tn5transposition, Tnp acts at two OE sequences (see Fig. 2 ). For IS50transposition, Tnp acts at one OE and one IE. (B) Schematic of Tnp primary structure. The locations of critical active-site residues are indicated with Xs. These include the DDE motif, the YREK signature sequence found in IS4family transposases, and K330. K330 is found in retroviral integrases and is similar to R found in other IS4family transposases. The black boxes marked with Cs locate domains involved in cisOE contacts. The open boxes marked with Ts locate domains involved in transOE contacts; TH is the transhairpin clamp contacts and TA is the transactive-site contacts. The shaded box is the location of the Tnp-Tnp dimer interface. Upward facing marks indicate hyperactive mutations. Downward facing marks are defective mutations caused by dimerization failure. (C) Tnp and Inh regulatory elements. The mRNAs that encode Tnp and Inh are programmed by two overlapping promoters. Two Dam methylation sites overlap the Tnp promoter −10 region. (D) End sequences. The nontransferred strands are presented for OE and IE with position 1 (5′) on the left. In addition, a hyperactive end sequence called the mosaic end (ME) is shown. The seven positions at which OE and IE differ are indicated in bold. The bold letters in the ME sequence indicate positions at which it has an OE-specific base in place of an IE base. Also shown are sites specific for host proteins: DnaA, Dam, and Fis.

Tn5transposes via a cut-and-paste conservative mechanism. Monomeric Tnp binds to the end sequences. Dimerization of Tnp and formation of transTnp-DNA contacts lead to formation of the synapse. Tnp (in the presence of Mg2+) cleaves the transposon DNA free from the donor backbone DNA through a three-step reaction.

Tnp-OE contacts. The structure data ( 17 ) were used to generate a model for the Tnp-OE contacts. Residues in roman type represent ciscontacts, and residues in italics represent transcontacts. Thick lines represent base-specific contacts in the major groove. Thin lines represent base-specific contacts in the minor groove. Dashed lines represent water-mediated interactions with bases. Phosphate interactions are also shown. This figure is based on the structure analysis by Douglas Davies and Scott Lovell.

Transposon-DBB cleavage and strand transfer. Tn5Tnp catalyzes cleavage through a hairpin intermediate that involves three sequential phosphoryl transfer reactions. In the first step, the transposon-transferred strand-donor DNA junction is nicked. The released 3′-OH of the transferred strand then attacks the opposite strand to form a hairpin intermediate. The hairpin is then nicked. Strand transfer then follows after target capture. The attack of the 3′-OH groups during strand transfer is staggered 9 bp apart.

Intramolecular transposition yielding nested deletions. Intramolecular transposition events yield two types of products depending on the precise orientation of the strand transfer reaction: deletion circles or inversion circles. The generation of deletion circles can be used as a tool for generating nested deletion families. The generation of two such deletions is pictured. The generation of inversion circles is not shown. Both types of products are convenient templates for sequencing. In the case pictured, the transposon has been constructed to have a target sequence, an origin of replication, and a selectable marker. Only deletion products from one side (carrying the origin and the selectable marker) are inherited after transformation.

Electroporation of preformed transposon-Tnp complexes. Tnp forms stable synaptic complexes with precleaved transposon DNA in the absence of Mg2+. Electroporation of these complexes into target cells yields transposition events. This technology avoids the need to produce Tnp in the target cells.

Random gene fusion technology. Two sequential transposition events are used to generate a random library of X-Y and Y-X fusions. The initial transposon is defined at its ends by IE sequences. Inside each IE sequence is an OE sequence in inverted orientation. The first transposition event uses the IE-specific hyperactive transposase sC7v2.0 ( 57 , 57a ) to make a library of inserts in gene X. The second transposition event uses the OE-specific hyperactive transposase EK-LP ( 91 ) to transpose the X and X′ containing transposons into gene Y