THE PLANT CELL, Vol 2, Issue 9 857-866, Copyright © 1990 by American Society of Plant Biologists
A Metal-Dependent DNA-Binding Protein Interacts with a Constitutive Element of a Light-Responsive Promoter
E. Lam, Y. Kano-Murakami, P. Gilmartin, B. Niner and N. H. Chua
Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021-6399
We have used DNase I footprinting to characterize nuclear factors that bind
to the light-responsive promoter of pea rbcS-3A, one member of the gene
family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase.
A sequence-specific binding activity, designated 3AF1, binds to an AT-rich
sequence present at the -45 region of the rbcS-3A promoter. A tetramer of
the 3AF1 binding site, designated as Box VI, can form multiple complexes
with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in
Box VI severely reduce DNA-protein complex formation in vitro. The
wild-type Box VI tetramer, but not the mutant tetramer, is active in
transgenic tobacco plants when placed upstream of the cauliflower mosaic
virus 35S promoter truncated at -90. These results correlate binding of
3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric
construct confers expression in diverse cell types and organs and its
activity is not dependent on light. By using the Box VI tetramer as a probe
to screen a cDNA expression library, we have obtained a putative cDNA clone
for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli
expressing the cDNA clone give sequence-specific complexes with Box VI. The
deduced amino acid sequence of the protein encoded by the cDNA contains two
stretches of about 100 residues that are 80% homologous. Moreover, in each
of the two repeats, there is an arrangement of histidines and cysteines,
which may be related to the two known types of zinc-finger motifs found in
many DNA-binding proteins. Consistent with the expectation that metal
coordination plays an important role in DNA binding by this protein, we
found that 1,10-phenanthroline can abolish the formation of DNA-protein
complexes. Interestingly, we found that the same treatment did not abolish
the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco.
These data indicate that the nuclear 3AF1 activity is likely due to
multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel
blot analysis shows that multiple transcripts homologous to this cDNA clone
are expressed in different tobacco organs.
