The order of the Tns coding regions of Tn7 and the position of the mutations isolated in TnsE. (A) Schematic representation of the Tn7 Tns coding regions and the relative positions of HindIII sites (H). The amino acid sequences of the C terminus of TnsD and the N terminus of TnsE are shown in the context of native Tn7 (Flores et al. 1990). The N-terminal protein sequences of two TnsE expression constructs (pJP117 and pJP118) are also shown. The peptide sequence obtained by sequencing purified TnsE and amino acid M37 are shown in bold and underlined (see text for details). (B) Schematic representation of the 538-amino-acid TnsE protein showing the approximate position of the high-activity mutations. (Circled residues) Mutations isolated as single mutations (see text for details).

The location and orientation of TnsABC+E-mediated transposition events in the E. coli chromosome and the conjugable F plasmid derivative pOX38-Gen. All of the transposition events were collected in NLC51 pOX38-Gen pCW15 (TnsABC) pJP103 (TnsE). Wild-type or mutant TnsE were expressed in pJP103; TnsE^WT (A), TnsE^A453V (B), TnsE^D523N (C), TnsE^{A453V/ G515R} (D), TnsE^{A453V/ D523N} (E). Arrows indicate the position of insertion events; numbering is based on the central base pair of the 5-bp duplication found with Tn7 transposition. Numbering of the chromosome and the pOX38-Gen plasmid follow the previously established conventions (Frost et al. 1994; Blattner et al. 1997). The orientation of insertion events is indicated by placement of the arrow inside or outside the circle according to the key. (Triangles) The chromosomal ter sites (terA-terJ); replisomes approaching from the flat side of the triangle will be halted. The position of the terminators is indicated by the central base pair of the 23-bp consensus ter binding site (Coskun-Ari and Hill 1997). The location of the bidirectional origin of chromosomal DNA replication, oriC, is indicated.

The location and orientation of transposition events mediated by mutant TnsE and the direction of DNA replication in the E. coli chromosome. The 50 insertion events promoted by the TnsE mutants that mapped to the E. coli chromosome in Fig. 2B–E are compiled. The position and orientation of oriC, the ter sites, and the TnsABC+E-mediated transposition events are as described in Fig. 2. (Blue dashed line) Replication events initiated at oriC in a clockwise orientation that terminate at the first properly oriented ter site, terC; (green dashed line) replication events initiated at oriC in a counter-clockwise orientation that terminate at the first properly oriented ter site, terA.

Gel mobility-shift assays indicate TnsE does not interact preferentially with single-stranded DNA. Gel mobility-shift assay is shown comparing TnsE^WT (lanes 2–4), TnsE^A453V/G515R (lanes 6–8), and TnsE^A453V/G483R (lanes 10–12). Increasing amounts of TnsE (0.06, 0.18, and 0.54 pmoles) were incubated with 0.05 pmoles of DNA substrate consisting of both single-stranded (ss) and double-stranded (ds) DNA for 30 min at 30°C and run on a 15% polyacrylamide gel (see Materials and Methods).

Certain DNA structures are preferred binding substrates for TnsE. Gel mobility-shift assay is shown comparing the ability of TnsE^WT and TnsE^A543V/G515R to bind various DNA structures. Each structure (0.08 pmoles) was incubated with no protein (Free DNA), or with 0.08, 0.24, or 0.72 pmoles of TnsE^WT (TnsE) or TnsE^A453V/G484R (TnsE*) for 30 min at 30°C and then run on a 6% polyacrylamide gel. The binding reactions contained a 50-fold excess of competitor DNA (see Materials and Methods). (=) Products found to comigrate when run in adjacent lanes; (*) the DNA strand that was 5′ end-labeled.