The double zinc finger domain and adjacent accessory domain from the transcription factor loss of zinc sensing 1 (loz1) are necessary for DNA binding and zinc sensing.
The Loz1 transcription factor from Schizosaccharomyces pombe plays an essential role in zinc homeostasis by repressing target gene expression in zinc-replete cells. To determine how Loz1 function is regulated by zinc, we employed a genetic screen to isolate mutants with impaired zinc-dependent gene expression and analyzed Loz1 protein truncations to map a minimal zinc-responsive domain. In the screen, we isolated 36 new loz1 alleles. 27 of these alleles contained mutations resulting in the truncation of the Loz1 protein. The remaining nine alleles contained point mutations leading to an amino acid substitution within a C-terminal double zinc finger domain. Further analysis of two of these substitutions revealed that they disrupted Loz1 DNA activity in vitro. By analyzing Loz1 protein truncations, we found that the last 96 amino acids of Loz1 was the smallest region that was able to confer partial zinc-dependent repression in vivo. This 96-amino acid region contains the double zinc finger domain and an accessory domain that enhances DNA binding. These results were further supported by the findings that MtfA, a transcription factor from Aspergillus nidulans that contains a related double zinc finger, is unable to complement loz1Δ, whereas a chimera of MtfA containing the Loz1 accessory domain is able to complement loz1Δ. Together, our studies indicate that the double zinc finger domain and adjacent accessory domain preceding zinc finger 1 are necessary for DNA binding and zinc-dependent repression.
From the Department of Molecular Genetics, the Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210.
Kate M
KM
Ehrensberger
From the Department of Molecular Genetics.
Mark E
ME
Corkins
the Department of Human Sciences, and.
Sangyong
S
Choi
From the Department of Molecular Genetics, the Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210the Department of Human Sciences, and bird.96@osu.edu.
Amanda J
AJ
Bird
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D004271:DNA, Fungal; C584539:Loz1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D015032:Zinc
Metal Homeostasis; Metallosensor; Transcription Repressor; Yeast Genetics; Zinc; Zinc Finger