University of Cologne
The structure of the DNA-binding domain of the Ac transposase
--Siegfried Feldmar and Reinhard Kunze
The ORFa protein, the putative transposase of the maize Ac transposon, has been over-expressed in E. coli. After renaturation, the protein has DNA binding properties indistinguishable from those of the ORFa protein expressed in insect cells. In order to characterize the DNA binding domain, we have constructed several mutant ORFa proteins (Feldmar, S and Kunze, R, EMBO J. 10:4003-4010, 1991). By deletion analysis the DNA binding domain was mapped to the N-terminal third of the protein, between amino acids 136 and 270. The protein segment between amino acids 159 and 207 carries many positively charged residues (14 basic versus one acidic residue). Of these, the basic residues between 190 and 200 are essential for the DNA binding reaction, as their substitution by uncharged amino acids results in the loss of DNA binding activity, whereas a similar exchange in neighboring regions does not affect it.
The amino acid sequence in this short region is indeed crucial for DNA-recognition. At a certain position not only a basic amino acid is required, but its identity is important, too. This was shown by exchanging histidine191 and arginine193. The resulting mutant is completely DNA binding deficient.
Immediately downstream of the DNA binding domain a region with weak
homology to a helix-loop-helix motif is located. This motif was found in
some DNA binding domains of other proteins. We have constructed three mutants
with partial or total deletions of the upstream and downstream helices,
respectively. All three mutants have virtually the same DNA binding properties
as the wildtype protein. We conclude that the potential helix-loop-helix
structure is not required for DNA binding. It might, however, have a different
function in vivo, since its mutation abolishes transposase function (see
report from Lütticke, Courage and Kunze, this issue).
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