Nuclear factors bind to hexamer motifs in subterminal sequences of Ac
--Heinz-Albert Becker and Reinhard Kunze
The transposable element Ac encoded ORFa protein can bind to AAACGG motifs in subterminal regions of the transposon but not to the 11bp terminal inverted repeats (Kunze et al., EMBO J. 8:3177, 1989). Host factors are known to be involved in the transposition mechanism of some other transposons. Therefore DNA-protein interaction studies were carried out looking for such factors.
Last year it was reported that three fragments of the Ac ends formed strong retarded complexes in electrophoretic mobility shift assays (EMSA) using crude nuclear extracts from kernels and pdIdC as an unspecific competitor. Fragment 1 (Ac-pos. 1-181), fragment 2 (Ac-pos. 4195-4419) and fragment 3 (Ac-pos. 4419-4565) showed homologous and heterologous competition in 10-fold excess of the non-labeled fragment.
To analyse the DNA-protein interactions in more detail all three Ac fragments were subjected to indirect DNaseI footprint analysis. Fragments 1 and 3 were used alternatively labeled at both strands. These experiments resulted in identification of a sequence motif which seems to be responsible for complex formation of the three Ac fragments. In regions where protected bases were clustered a hexamer motif could be identified. The sequence is GxTAAA, where x is a G in 4, and A in 2 cases and a T in one case. A GCTAAA doesn't occur in Ac. The GxTAAA motifs lie in between the AAACGG hexamers known to be recognized by the Ac ORFa protein. The hexamer is the only consensus sequence among the protected sites. Under the conditions applied the protected regions containing the GxTAAAs were the only ones in the three Ac fragments. No protection of the terminal 11bp inverted repeats was detectable. The hexamer is found 3 times in the first 181bp of Ac and 6 times in the last 369bp. At 7 positions protected bases can be seen. One position shows no clear protection and the last hasn't been analyzed so far.
For efficient DNA-protein complex formation in EMSAs more than one hexamer seems to be necessary per fragment. Each of the three strongly complexed Ac fragments contains more than one motif. When fragment 1 is split at Ac pos. 75 the fragment pos. 76-181 is strongly complexed. It still contains 2 GGTAAAs. Fragment 1-75, which has only one hexamer, is only very weakly complexed.
These observations are in coincidence with the use of isolated host factor motifs. The hexamer at Ac pos. 4533 was subcloned with two upstream and downstream bases as a 14bp oligonucleotide with Sau3A spacers in vector pUC19. A copy number of four seems to be necessary for efficient and specific complex formation in vitro. It can compete in 10-fold excess complex formation of the Ac fragments 1 and 3, but the reverse competition experiment wasn't successful. The subcloned GGTAAA motif seems to be stronger complexed than the Ac fragments with the different GxTAAAs. This observation and the fact that one of the 9 GxTAAAs in the Ac ends showed no clear protection leads to the assumption that neighbour bases might have an additional influence on the strength of complex formation.
So far there is no evidence for a role of the observed DNA-protein interactions in the transposition mechanism of Ac.
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