Figure 1

Tn5, transpososome structure and transposon substrates. (A) The organization of Tn5 (5.8 kb) is shown. The outside (OE) and inside ends (IE) of Tn5 (half arrows) contain determinants for transposase binding. IS50 Right encodes transposase and other ORF's are shown (thin arrows show the corresponding transcription unit). Flanking donor DNA is represented as orange rectangles. Below the schematic of Tn5 an illustration of the basic transposon end fragment (53 basepair) used for in vitro binding studies is shown. In addition, the sequence of the terminal 19 nucleotides, as well as 3 flanking nucleotides, of the non-transferred strand of each of the four transposon end substrates used in this work is shown (ME - mosaic end). Basepair changes between the ME and mutant forms of the ME (ME 3 and ME 8/9) are in blue. Basepair differences between OE and ME sequences are in red. The DnaA binding site is also shown. Potential H-NS binding sites inferred from hydroxyl radical footprinting are also shown and identified as either sites 1, 2 or 3. (B) Sites 1, 2 and 3 represent potential H-NS binding sites of the H-NS-Tn5 transpososome as described in (A); the light and dark pink spheres placed in the structural model of the Tn5 transpososome represent positions of weak and strong protection against hydroxyl radical attack, respectively. (C) Schematic of plasmids used for mating out assays. The plasmid on the left contains the mini-Tn5-Kan element with arrows in boxes depicting the end sequence (ME - pDH626, OE - pDH689, ME 3 - pDH685, or ME 8/9 - pDH660) and the thick blue arrow indicates the kanamycin resistance gene. The plasmid on the right encodes transposase (MA56) (purple arrow) under control of its native promoter. Black boxes represent the origins of replication, pMB1 and p15A; Ap^R, Cm^R and Tet^R encode resistance genes for ampicillin, chloramphenicol and tetracycline, respectively. Note that the non-mutated ME is referred to as the wild type (WT) ME in this work.