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. Nietosotelo et al., 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, The Plant cell, 6(2), 1994, pp. 287-301
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.