Functional independence of the two cysteine-rich activation domains in the yeast Mac1 transcription factor.
Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the (264)CXC[(X)(4)]CXC[(X)(2)]C[(X)(2)][H(279)]C1 motif. The lone exception was a L260S substitution. Two additional MAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.
Keller
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G
University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.
Gross
C
C
Kelleher
M
M
Winge
D R
DR
D000595:Amino Acid Sequence; D003545:Cysteine; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D005656:Fungal Proteins; C084954:MAC1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003545:Cysteine