The Ac transposase (TPase) binds in vitro to short sequence motifs (AAACGG) which occur in multiple copies but different arrangements in both ends of Ac and Ds. By testing a variety of subterminal Ds mutants for transpositional competence in vivo (see report of Shivani Chatterjee and Peter Starlinger) it was found that regions free of AAACGG motifs in the 5'-end of Ds are also important for transposition. However, these regions contain motifs which are similar to AAACGG. We have begun to analyze the structural and sequence requirements for specific binding of TPase to DNA in vitro. We performed gel shift assays with the N-terminally truncated TPase(103-807) and radiolabelled plasmid fragments containing various oligonucleotides in different copy numbers, distances and orientations.
When using tandem arrays of AAACGGs as probe, two copies are very weakly complexed, whereas the amount of stable TPase/DNA-complex increases dramatically with higher copy numbers. It turned out that a DNA-unit length of 5 nucleotides is sufficient for complexation. Tandem arrays of six AAACG-, AACGG- and ACGGG-motifs are efficiently bound by the TPase. In contrast, the tetramer ACGG and several motifs with base substitutions in different positions (AGCGG, CGCGG, AAAGGG, AAACTG) are not complexed. The arrangement of motifs in opposite orientations leads to a strongly reduced but not completely abolished binding efficiency. In summary, these experiments show that DNA-recognition by the TPase is not restricted to a single DNA motif. A WCG-motif repeated in tandem several times and interrupted by two or three nucleotides long spacers (longer spacers have not yet been analyzed) seems to be sufficient for specific binding by the TPase. Individual binding motifs are - except for the central CG-dinucleotide - not palindromic, and tandem arrays of binding motifs result in strongly increased amounts and/or stability of TPase/DNA-complexes. These observations could indicate that individual TPase molecules interact non-symmetrically with each other, forming oligomers variable in size.
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