Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA.
A change of a universally conserved leucine to valine in the DNA-binding domain of the GATA factor AreA results in inability to activate some AreA-dependent promoters, including that of the uapA gene encoding a specific urate-xanthine permease. Some other AreA-dependent promoters become able to function more efficiently than in the wild-type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild-type and mutant forms of AreA. In vitro binding studies of wild-type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild-type and mutant AreA-DNA complexes derived from the atomic coordinates of the GATA-1-AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA-binding sites, is sufficient to ensure activation of transcription of the uapA gene.
Ravagnani
A
A
Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, London, UK.
Gorfinkiel
L
L
Langdon
T
T
Diallinas
G
G
Adjadj
E
E
Demais
S
S
Gorton
D
D
Arst
H N
HN
Scazzocchio
C
C
D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D026901:Membrane Transport Proteins / Q000235:genetics*; D008958:Models, Molecular; D015394:Molecular Structure; D010641:Phenotype; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014867:Water; D016335:Zinc Fingers
C079728:AreA protein, Aspergillus nidulans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C083852:UAPA protein, Aspergillus nidulans; D014867:Water