The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex.

Budding yeast HAP2 is required in concert with HAP3 and HAP4 to form a heterotrimeric CCAAT-binding transcriptional activation complex at the UAS2 element of CYC1. Functional homologs of HAP2 and HAP3 have been conserved in HeLa cells where HAP2 activity corresponds to a chromatographic fraction designated CP1B. Here, we describe deletion and codon insertion mutagenesis of the Saccharomyces cerevisiae HAP2 subunit. DNA binding was assessed both in vitro and in vivo whereas subunit association and transcriptional activation were analyzed in vivo by using a bifunctional lexA-HAP2 fusion. The results indicate that the 265-amino-acid HAP2 protein contains an "essential core" of 65 amino acids, which is entirely sufficient for assembly and DNA binding of the HAP2/3/4 complex. The essential core can be divided further into a region of 44 amino acids, which is sufficient for subunit association, and a region of 21 amino acids, which is required specifically for DNA recognition. The remainder of the HAP2 protein is dispensable. The roles of HAP3 and HAP4 in the heterotrimeric complex are also assessed by using lexA fusions and a fusion of the GAL4 acidic activation domain to HAP2, which partially bypasses a deletion of HAP4 but not HAP3. These data indicate that subunits HAP2 and HAP3 are primarily responsible for site-specific DNA binding by the complex, whereas the HAP4 subunit provides the primary transcriptional activation domain. A model for the function of this regulated transcriptional activation complex is presented.

[1]  Tsonwin Hai,et al.  Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. , 1989, Genes & development.

[2]  L. Guarente,et al.  N-terminal deletions of a mitochondrial signal sequence in yeast. Targeting information of delta-aminolevulinate synthase is encoded in non-overlapping regions. , 1989, The Journal of biological chemistry.

[3]  D. Lockshon,et al.  MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer , 1989, Cell.

[4]  E. A. O'neill,et al.  The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain , 1989, Cell.

[5]  T. Kouzarides,et al.  Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding , 1989, Nature.

[6]  Y. Jan,et al.  Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence , 1989, Cell.

[7]  M. Scott,et al.  The structure and function of the homeodomain. , 1989, Biochimica et biophysica acta.

[8]  J. Berg.  DNA binding specificity of steroid receptors , 1989, Cell.

[9]  R. Müller,et al.  Two functionally different regions in Fos are required for the sequence-specific DNA interaction of the Fos/Jun protein complex , 1989, Nature.

[10]  David Baltimore,et al.  A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins , 1989, Cell.

[11]  B. Matthews,et al.  The helix-turn-helix DNA binding motif. , 1989, The Journal of biological chemistry.

[12]  R. Rothstein.  18 – One-Step Gene Disruption in Yeast , 1989 .

[13]  R. Tjian,et al.  Analysis of Sp1 in vivo reveals mutiple transcriptional domains, including a novel glutamine-rich activation motif , 1988, Cell.

[14]  M. Ptashne.  How eukaryotic transcriptional activators work , 1988, Nature.

[15]  C. S. Parker,et al.  Isolation of the gene encoding the S. cerevisiae heat shock transcription factor , 1988, Cell.

[16]  S. McKnight,et al.  The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. , 1988, Science.

[17]  L. Guarente,et al.  Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex. , 1988, Science.

[18]  P. Sharp,et al.  Human CCAAT-binding proteins have heterologous subunits , 1988, Cell.

[19]  P. Sharp,et al.  A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable , 1988, Cell.

[20]  L. Guarente,et al.  Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site , 1988, Molecular and cellular biology.

[21]  L. Guarente,et al.  The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts , 1988, Molecular and cellular biology.

[22]  R. Brent,et al.  DNA-bound Fos proteins activate transcription in yeast , 1988, Cell.

[23]  Steven Hahn,et al.  Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner , 1987, Cell.

[24]  L. Guarente,et al.  Constitutive expression of the yeast HEM1 gene is actually a composite of activation and repression. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Jun Ma,et al.  Deletion analysis of GAL4 defines two transcriptional activating segments , 1987, Cell.

[26]  L. Guarente,et al.  Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator , 1987, Molecular and cellular biology.

[27]  Aaron Klug,et al.  ‘Zinc fingers’: a novel protein motif for nucleic acid recognition , 1987 .

[28]  G. Rose,et al.  Loops in globular proteins: a novel category of secondary structure. , 1986, Science.

[29]  L. Guarente,et al.  Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae , 1985, Molecular and cellular biology.

[30]  R. Brent,et al.  A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor , 1985, Cell.

[31]  F. Barany.  Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Artavanis-Tsakonas,et al.  opa: A novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster , 1985, Cell.

[33]  L. Guarente,et al.  Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae , 1984, Cell.

[34]  R. Sauer,et al.  Protein-DNA recognition. , 1984, Annual review of biochemistry.

[35]  L. Guarente,et al.  Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site , 1983, Cell.

[36]  J. Ferguson,et al.  mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. , 1983, The Journal of biological chemistry.

[37]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[38]  G. Fink,et al.  Methods in yeast genetics , 1979 .