In vitro analysis of the Ac encoded ORFa protein and mutant forms after heterologous expression
--Siegfried Feldmar and Reinhard Kunze
The transposable element Ac is transcribed in the form of one 3.5kb long transcript. The first AUG codon of the Ac mRNA opens an 807 amino acid long open reading frame called ORFa. A corresponding protein with an apparent molecular weight of about 112kD is found in nuclear extracts by western blot analysis.
It was shown that the ORFa protein expressed in insect cells has DNA binding activity; the recombinant ORFa protein binds specifically to several subterminal fragments from both ends of Ac. The recombinant protein also binds to DNA fragments containing concatemers of an AAACGG motif that is repeated several times at both ends of Ac (Kunze et al., EMBO J. 8:3177, 1988). In order to identify the structural basis for the site specific DNA binding of the Ac ORFa protein we began to express the intact and mutant forms of the protein in E. coli.
The recombinant proteins were accumulated in an insoluble and inactive form in the bacteria. The protein aggregates were recovered by cell breakage and low speed centrifugation. After solubilization in guanidinium chloride and subsequent refolding by dilution the soluble fraction of the proteins was analyzed for DNA binding activity using the gel mobility shift assay.
We could demonstrate that renatured wildtype protein has DNA binding properties comparable to the ORFa protein expressed in insect cells. From these studies we concluded that the wildtype ORFa protein can be reactivated after denaturation in guanidinium chloride. Protein modifications that might occur in higher eukaryotic cells are not essential for site specific DNA binding.
To roughly localize the region of ORFa necessary for specific DNA binding, a series of N-terminal and C-terminal deleted forms of the protein were tested for their ability to bind specifically to the Ac5'-end and the AAACGG motif. We found the 537 amino acids can be removed from the C-terminus without disrupting the capacity to complex specifically the ORFa protein target sites. The N-terminus of the protein is not required for DNA-binding, too. A truncated version of the ORFa protein, having lost 135 amino acids from the N-terminus, still has full DNA-binding activity.
From these studies we conclude that a DNA binding domain of the protein resides somewhere between residues 136 and 370. The N-terminal half of this segment has a rather basic character, whereas the C-terminal half reveals a weak homology to the helix-loop-helix motif. Such motifs are found in the DNA-binding domains of some proteins (Murre et al., Cell 56:777, 1989).
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