Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence.
Transposon Tn10 inserts preferentially into particular "hotspots" that have been shown by sequence analysis to contain the symmetrical consensus sequence 5'-GCTNAGC-3'. This consensus is necessary but not sufficient to determine insertion specificity. We have mutagenized a known hotspot to identify other determinants for insertion into this site. This genetic dissection of the sequence context of a protein binding site shows that a second major determinant for Tn10 insertion specificity is contributed by the 6-9 base pairs that flank each end of the consensus sequence. Variations in these context base pairs can confer variations of at least 1000-fold in insertion frequency. There is no discernible consensus sequence for the context determinant, suggesting that sequence-specific protein-DNA contacts are not playing a major role. Taken together with previous work, the observations presented suggest a model for the interaction of transposase with the insertion site: symmetrically disposed subunits bind with specific contacts to the major groove of consensus-sequence base pairs, while flanking sequences influence the interaction through effects on DNA helix structure. We also show that the determinants important for insertion into a site are not important for transposition out of that site.
Bender
J
J
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Kleckner
N
N
D001483:Base Sequence; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004271:DNA, Fungal; D006639:Histidine; D009713:Nucleotidyltransferases; D019895:Transposases