Maize Genetics Cooperation Newsletter vol 84 2010
Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of authors.
Florida State University
A recombinant C-terminal fragment of maize Initiator Binding Protein 2 (IBP2) binds to telomere-repeat DNA in vitro.
The maize (Zea mays L.) initiator-binding protein2 (IBP2) gene is known to encode a protein, IBP2, that binds to the initiator (inr) element in the Shrunken1 promoter (Lugert and Werr 1994; Plant Mol Biol, 25, 493–506). The duplicate genes IBP1 and IBP2 were initially identified in ligand-binding screens using cDNA expression libraries. The binding of IBP to the transcription start site inr element was confirmed by footprint and band-shift assays. We and others have observed that IBP1 and IBP2 also resemble telomere DNA-binding proteins from other plant species. We investigated therefore whether the IBP2 protein exhibits telomere DNA-binding activity.
Using Electrophoretic Mobility Shift Assay (EMSA) we found that a C-terminal sublcone of the IBP2 protein (rIBP2 Myb-Q, AA 579-667, GenBank GU080214), binds telomeric repeat DNA in vitro (Fig. 1, arrows indicate band shifts). We also found that rIBP2 Myb-Q binds strongly to double stranded telomere repeat sequences with three or four tandem repeats, but only weakly to sequences with two (Fig. 2, arrows indicate band shifts). Furthermore, we found no evidence of binding with only one telomere repeat (5Õ TTTAGGG 3Õ) sequence. Single point mutations (T1:A and G6:T) in the three-repeat sequence diminished, but did not entirely abolish, binding activity (via competition EMSA assays, data not shown).
In order to compare the binding of rIBP2 Myb-Q to telomere vs. initiator DNA, we carried out filter binding assays. In this experiment, total protein extract from E coli cultures expressing rIBP2 Myb-Q (IPTG+) were incubated with nitrocellulose filters. These filters were then incubated with radiolabeled oligonucleotide probes corresponding to telomere DNA or Sh1 initiator DNA (Fig. 3). From these experiments, we found that the rIBP2 Myb-Q showed very weak binding to the initiator sequence of the Shrunken1 promoter. These findings confirm the prediction that the maize IBP protein (IBP1 or IBP2) has telomere-DNA-binding activity. Consequently, maize IBP1/2 may reside at maize telomeres, raising the intriguing possibility of dual functions for IBP, telomeric and transcriptional.