THE PLANT CELL, Vol 6, Issue 2 287-301, Copyright © 1994 by American Society of Plant Biologists
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
J. Nieto-Sotelo, A. Ichida and P. H. Quail
University of California-Berkeley/United States Department of Agriculture, Plant Gene Expression Center, 800 Buchanan Street, Albany, California 94710
Phytochrome-imposed down-regulation of the expression of its own
phytochrome A gene (PHYA) is one of the fastest light-induced effects on
transcription reported in plants to date. Functional analysis of the oat
PHYA3 promoter in a transfection assay has revealed two positive
elements,PE1 and PE3, that function synergistically to support high levels
of transcription in the absence of light. We have isolated a rice cDNA
clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1
element. We tested the selectivity of the pR4-encoded DNA binding activity
using linker substitution mutations of PE1 that are known to 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 behavior expected of
positive factor 1 (PF1), the presumptive nuclear transcription 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 high-mobility group I-Y (HMG I-Y) proteins.
In addition, PF1 contains an 11-amino acid-long hydrophobic region
characteristic of HMG I proteins, its N-terminal region shows strong
similarities to a pea H1 histone 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 quantitative gel shift analyses showed that PF1 binds
preferentially to the PE1 element but also at lower affinity to two other
AT-rich regions upstream of PE1. This feature is consistent with the
binding characteristics 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
containing both histone H1 domains and A-T hook DNA binding domains.
