Tn 5 /IS 50 target recognition

Abstract

This communication reports an analysis of Tn5͞IS50 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 Tn5͞IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5͞IS50 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 Tn5͞IS50 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.

From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways

“…Libraries of 2 ϫ 10 5 independent insertion mutants were generated in E. coli strains MG1655 and DH10B using Tn5-based EZ::TNϽKAN-2ϾTnp transposome (Epicentre Technologies) as a transposon delivery system. It utilizes a mutant hyperactive form of the Tn5 transposase, which has been reported to have low insertion site specificity (41,64). In our study EZ::TN transposase was found to be sufficiently random to yield 1 to 14 independent insertions within a majority of E. coli genes (for example, see Fig.…”

Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus

“…Afterward, transposon mutagenesis was performed on pDD365 using the EZ::TN In-Frame Linker Insertion Kit (Epicenter Technologies). This method relies on the ability of a modified 1000-bp Tn5 transposon element-coupled to transposase to insert itself randomly into target DNA in vitro (81,84). Transposon events are identified by kanamycin resistance.…”

Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

“…To test this, two transposon-containing plasmids were used that differed only by the presence of outside end sequences in pRZTL1 and mosaic end sequences in pRZTL4. No significant differences were noted in transposition frequencies between the K40Q mutant and the hyperactive EKLP parent transposase with either form of the transposons; however, the transposition frequency using mosaic ends was about 10ϫ greater than using outside ends for both transposases (Table I, columns [3][4][5][6]. Based on these data, the 100ϫ difference in activity between these two transposase molecules observed in the in vivo mating out assay is due to a process that occurs in vivo not measured by the in vitro assay.…”

“…1 below). Genetic engineering of Tn5 transposase to generate a hyperactive form (EKLP) 1 and the optimization of the transposon end sequences allow many of the individual steps of the Tn5 transposition mechanism to be studied in vitro (1)(2)(3)(4)(5)(6). The hyperactive EKLP form of Tn5 transposase contains the mutations E54K, M56A, and L372P (4).…”