Nucleic Acids Research, 1994, Vol. 22, No. 5 773-779
© 1994
MOLECULAR BIOLOGY |
Transposase A binding sites in the attachment sites of bacteriophage Mu that are essential for the activity of the enhancer and A binding sites that promote transposition towards Fpro-lac
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University Einsteinweg 55, 2300 RA Leiden, The Netherlands
*To whom correspondence should be addressed
Received December 8, 1993. Revised February 7, 1994. Accepted February 7, 1994.
In this paper we determine which of the A binding sites In the attachment sites of phage Mu are required for the stimulatory activity of the transpositional enhancer (IAS). For this purpose the transposition frequencies of mini-Mu's with different truncated attachment sites to an Ftet target were measured both in the presence and the absence of the IAS. The results show that in our in vivo assay the L3 and R3 sites are dispensable for functioning of the IAS. An additional deletion of L2 or R2 however abolishes the stimulating activity of the enhancer suggesting an interaction between A molecules bound to these sites and the IAS. The residual transposition activity of a IAS-containing mini Mu in which R2 (and R3) are deleted is much lower than the activity of the comparable construct without the IAS. This means that in the absence of R2 the IAS is Inhibiting transposition. Such an inhibition is not observed when L2 (and L3) are deleted. This suggests that the IAS interacts with the attachment sites In an ordered fashion, first with affL and then with attR. Furthermore we show that mini-Mu transposition is enhanced when Fpro-lac is used as a target instead of Ftet. We show that this elevated transposition Is dependent on the Mu A binding sites L2.L3 and R2. These sequences could possibly mediate an Interaction between the mlnl-Mu plasmid and sequences present on Fpro-lac.