PF1 - AN A-T HOOK-CONTAINING DNA-BINDING PROTEIN FROM RICE THAT INTERACTS WITH A FUNCTIONALLY DEFINED D(AT)-RICH ELEMENT IN THE OAT PHYTOCHROME A3 GENE PROMOTER

Phytochrome-imposed down-regulation of the expression of its own phyto chrome A gene (PHYA) is one of the fastest light-induced effects on tr anscription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elem ents, PE1 and PE3, that function synergistically to support high level s of transcription in the absence of light. We have isolated a rice cD NA clone (pR4) encoding a DNA binding protein that binds to the AT-ric h PE1 element. We tested the selectivity of the pR4-encoded DNA bindin g activity using linker substitution mutations of PEI that are known t o disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the be havior expected of positive factor 1 (PF1), the presumptive nuclear tr anscription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in h igh-mobility group I-Y (HMG I-Y) proteins. In addition, PF1 contains a n 11-amino acid-long hydrophobic region characteristic of HMG I protei ns, its N-terminal region shows strong similarities to a pea H1 histon e sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and qua ntitative gel shift analyses showed that PF1 binds preferentially to t he PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characte ristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins contai ning both histone H1 domains and A-T hook DNA binding domains.