Functional characterization of the iron-regulatory transcription factor Fep1 from Schizosaccharomyces pombe.
In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5'-(A/T)GATAA-3', found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity approximately 5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1(+) mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5'-(A/T)GATAA-3' reflects a much lower affinity that correlates with failure to repress fio1(+) gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis.
Pelletier
Benoit
B
Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Trott
Amy
A
Morano
Kevin A
KA
Labbé
Simon
S
D000409:Alanine / Q000737:chemistry; D000483:Alleles; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D050980:GATA Transcription Factors; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C459859:Fep1 protein, S pombe; D050980:GATA Transcription Factors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA; D007501:Iron; D007930:Leucine; D003545:Cysteine; D000409:Alanine