Characterization of a DNA-binding protein that interacts with 5' flanking regions of two fruit-ripening genes

The E4/E8 binding protein (E4/E8BP) interacts with sequences in the 5' flanking regions of two genes, E4 and E8,that are coordinately regulated by ethylene during tomato fruit ripening. The DNA-binding activity of this protein increases during fruit ripening, and it may play a role in regulation of these genes. To begin to understand the function of this protein, a cDNA has been isolated that encodes a protein, E4/E8BP-1, with DNA-binding specificity similar to that of E4/E8BP. This DNA-binding protein is closely related to a DNA binding protein from tobacco, 3AF1,that interacts with the promoter of the pea rbcS-3A gene. A repeated domain was identified within the predicted 3AF1 amino acid sequence, which includes a series of histidines and cysteines, suggestive of zinc binding, and this repeat is conserved in E4/E8BP-1. Interaction of both E4/E8BP-1 and nuclear extracts from ripening fruit with the E8 recognition sequence is sensitive to 1,10-phenanthroline, indicating that a metal is required for binding of both the native and recombinant proteins. The mRNA for E4/E8BP-1 is moderately abundant in fruit, and increases slightly during fruit ripening, consistent with a role in fruit ripening. A truncated version of E4/E8BP-1 was able to transactivate the E4 promoter in transient assay, demonstrating that this DNA-binding protein can interact with the E4 promoter in vivo to enhance gene transcription.