CHARACTERIZATION OF A DNA-BINDING PROTEIN THAT INTERACTS WITH 5'-FLANKING REGIONS OF 2 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 regula ted by ethylene during tomato fruit ripening. The DNA-binding activity of this protein increases during fruit ripening, and it may play a ro le in regulation of these genes. To begin to understand the function o f this protein, a cDNA has been isolated that encodes a protein, E4/E8 BP-1, with DNA-binding specificity similar to that of E4/E8BP. This DN A-binding protein is closely related to a DNA binding protein from tob acco, 3AF1,that interacts with the promoter of the pea rbcS-3A gene. A repeated domain was identified within the predicted 3AF1 amino acid s equence, which includes a series of histidines and cysteines, suggesti ve of zinc binding, and this repeat is conserved in E4/E8BP-1. Interac tion of both E4/E8BP-1 and nuclear extracts from ripening fruit with t he E8 recognition sequence is sensitive to 1,10-phenanthroline, indica ting that a metal is required for binding of both the native and recom binant proteins. The mRNA for E4/E8BP-1 is moderately abundant in frui t, and increases slightly during fruit ripening, consistent with a rol e in fruit ripening. A truncated version of E4/E8BP-1 was able to tran sactivate the E4 promoter in transient assay, demonstrating that this DNA-binding protein can interact with the E4 promoter in vivo to enhan ce gene transcription.