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Abstract
Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA
elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling
the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated
by a number of small cyclic peptides, known as siderophores. The siderophore synthesis
pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed
partial constitutive synthesis of siderophore. In the research presented here, the
negative function of SRE was further confirmed by a heterokaryon test and by gene
complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed
that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation
of sre gene expression appears possible because the sre gene promoter itself contains
GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions
in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed
that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding.
This feature is different from that found with the vertebrate two zinc finger GATA
factors. Invivo tests, accomplished by transforming the mutant sre genes into sre
rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained
its function under low-iron conditions. In contrast, these mutant SRE proteins fail
to function under high-iron conditions. Our results predict the presence of other
positive or negative regulators of the siderophore synthetic pathway.