Quantitation of oligohistidine fusion proteins using<sup>63</sup>Ni<sub>2+</sub>chelation

Ravagnani, Adriana; Arst, Herbert N.

doi:10.1093/nar/23.15.3069

Cited by 3 publications

(3 citation statements)

References 7 publications

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“…Expression, purification and quantitation of wild‐type and mutant fusion proteins were described in Langdon et al . (1995) and Ravagnani and Arst (1995).…”

Section: Methodsmentioning

confidence: 98%

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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

Ravagnani

Gorfinkiel

Langdon

et al. 1997

EMBO J

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161

166

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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.

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“…Expression, purification and quantitation of wild‐type and mutant fusion proteins were described in Langdon et al . (1995) and Ravagnani and Arst (1995).…”

Section: Methodsmentioning

confidence: 98%

“…Expression, purification and quantitation of wild-type and mutant The dUTP interference was adapted from Pu and Struhl (1992). A PCR probe was prepared as described above, including 30 μM dUTP fusion proteins were described in Langdon et al (1995) and Ravagnani and Arst (1995).…”

Section: Plasmid Construction Expression and Purification Of The Olig...mentioning

confidence: 99%

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

Ravagnani

Gorfinkiel

Langdon

et al. 1997

EMBO J

Self Cite

161

166

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“…The ability of the AREA DBD to bind specifically to GATA sequences is well documented (24,46,52,58,60,64,65,76,77). GATA sequences in at least two promoters have been shown to be physiologically relevant to AREA control (46,65).…”

Section: Introductionmentioning

confidence: 99%

Mutational Analysis of AREA, a Transcriptional Activator Mediating Nitrogen Metabolite Repression in Aspergillus nidulans and a Member of the “Streetwise” GATA Family of Transcription Factors

Wilson

Arst

1998

Microbiol Mol Biol Rev

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117

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SUMMARY The transcriptional activator AREA is a member of the GATA family of transcription factors and mediates nitrogen metabolite repression in the fungus Aspergillus nidulans. The nutritional versatility of A. nidulans and its amenability to classical and reverse genetic manipulations make the AREA DNA binding domain (DBD) a useful model for analyzing GATA family DBDs, particularly as structures of two AREA-DNA complexes have been determined. The 109 extant mutant forms of the AREA DBD surveyed here constitute one of the highest totals of eukaryotic transcription factor DBD mutants, are discussed in light of the roles of individual residues, and are compared to corresponding mutant sequence changes in other fungal GATA factor DBDs. Other topics include delineation of the DBD using both homology and mutational truncation, use of frameshift reversion to detect regions of tolerance to mutational change, the finding that duplication of the DBD can apparently enhance AREA function, and use of the AREA system to analyze a vertebrate GATA factor DBD. Some major points to emerge from work on the AREA DBD are (i) tolerance to sequence change (with retention of function) is surprisingly great, (ii) mutational changes in a transcription factor can have widely differing, even opposing, effects on expression of different structural genes so that monitoring expression of one or even several structural genes can be insufficient and possibly misleading, and (iii) a mutational change altering local hydrophobic packing and DNA binding target specificity can markedly influence the behavior of mutational changes elsewhere in the DBD.

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Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

et al. 1996

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In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

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Quantitation of oligohistidine fusion proteins using⁶³Ni₂₊chelation

Cited by 3 publications

References 7 publications

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

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

Mutational Analysis of AREA, a Transcriptional Activator Mediating Nitrogen Metabolite Repression in Aspergillus nidulans and a Member of the “Streetwise” GATA Family of Transcription Factors

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

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Quantitation of oligohistidine fusion proteins using63Ni2+chelation

Cited by 3 publications

References 7 publications

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

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

Mutational Analysis of AREA, a Transcriptional Activator Mediating Nitrogen Metabolite Repression in Aspergillus nidulans and a Member of the “Streetwise” GATA Family of Transcription Factors

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

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Quantitation of oligohistidine fusion proteins using⁶³Ni₂₊chelation