The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis.
The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N'-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1-1 and P-491 to L in urbs1-3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1-minus phenotype. The third NTG urbs1 mutant (urbs1-2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1-minus phenotype. A second frame shift mutation was identified in urbs1-2 and is necessary for the urbs1-minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.
An
Z
Z
Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706, USA.
Zhao
Q
Q
McEvoy
J
J
Yuan
W M
WM
Markley
J L
JL
Leong
S A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins; D005800:Genes, Fungal; D007501:Iron / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D017262:Siderophores / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014588:Ustilago / Q000235:genetics / Q000502:physiology*; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D017262:Siderophores; D014157:Transcription Factors; C083724:urbs1 protein, Ustilago maydis; D007501:Iron; D006899:Mixed Function Oxygenases; C059419:ornithine N5-oxygenase