THE ORGANIZATION OF THE OUTSIDE END OF TRANSPOSON TN5

The end sequences of the IS50 insertion sequence are known as the outs ide end (OE) and inside end. These complex ends are related but nonide ntical 19-bp sequences that serve as substrates for the activity of th e Tn5 transposase. Besides providing the binding site of the transposa se, the end sequences of a transposon contain additional types of info rmation necessary for transposition. These additional properties inclu de but are not limited to host protein interaction sites and sites tha t program synapsis and cleavage events. In order to delineate the prop erties of the IS50 ends, the base pairs involved in the transposase bi nding site have been defined. This has been approached through perform ing a variety of in vitro analyses: a hydroxyl radical missing-nucleos ide interference experiment, a dimethyl sulfate interference experimen t, and an examination of the relative binding affinities of single-sit e end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, includi ng positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.