Tn5/IS50 target recognition

Abstract

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

Figure 1

Plasmid pRZTL1 (described in more detail in ref. 23). The two solid squares are the OE sequences of Tn5 which define the transposable element. The shorter DNA sequence between the ends encoding kanamycin resistance is a donor backbone. Along with a p15A origin of replication and the Cam^R gene, the transposon contains a promoterless Tet^R gene next to one OE. Tetracycline resistance can be activated after transposition in the proper orientation into a unit of transcription. Since the same Km^R gene was used as a target in two other in vivo systems, we were able to compare all sets of data. The location into which the synthetic targets were cloned is indicated.

Figure 2

In vitro Tn5 insertions in the Cm^R gene of pRZTL1. The Upper map indicates all of the in vitro-generated Tn5 insertions in the Cm^R gene. In many cases independent insertions (especially those in sites hit more than once) frequently are located in close, overlapping positions. Two examples of insertion clusters are shown, with the bars identifying the middle base pair of the 9-bp SDR. Solid bars represent insertions with five-letter shifts. Other insertions, shown by open bars, in some cases fall into alternative groups demonstrating 5- or 10-letter shifts. Below the text, targets in the area demonstrating a 5-bp periodicity are underlined to show actual 9-bp targets.

Figure 3

Occurrences of particular distances between insertion points within 25 bp in plasmid pRZTL1. The abundance of insertion sites (found two or more times) located within 25 bp in a clockwise direction (Fig. 1) was determined relative to each site.

Figure 4

Synthetic target DNAs checked for efficiency and precision of integration. Different linker DNAs designed as described in Results were inserted into the EcoRI-NcoI large fragment of pRZTL1, replacing a part of the Cm^R gene (see Fig. 1). Insertions into the synthetic target can activate Tet^R as found for the original pRZTL1 plasmid. Middle letters of predicted target sites are highlighted. Actual target sites are shown below the text, with the number of occurrences indicated. For all three regions, only two cases did not fit the predicted pattern.

Figure 5

A model for target recognition by the transposase–OE synaptic complex. Transposase is proposed to form a filament-like multimeric structure on target DNA. Within the filament, one pair of transposase monomers that formed a synaptic complex with two OE sequences makes the strand transfer. A tendency of transposase to form a multimer presumably increases specificity of integration because of cooperative DNA recognition.