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L Guarente

Publications and source records attributed to L Guarente.

At least 19 recordsLinked to original sources
PubMed1992

Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains.

We have devised a genetic strategy to isolate the target of acidic activation domains of transcriptional activators based on toxicity in yeast cells of the chimeric activator, GAL4-VP16. Toxicity required the integrity of both the VP16 acidic activation domain and the GAL4 DNA-binding domain, suggesting that inhibition resulted from trapping of general transcription factors at genomic sites. Mutations that break the interaction between GAL4-VP16 and general factors would alleviate toxicity and identify transcriptional adaptors, if adaptors bridged the interaction between activators and general factors. We thus identified ADA1, ADA2, and ADA3. Mutations in ADA2 reduced the activity of GAL4-VP16 and GCN4 in vivo. ada2 mutant extracts exhibited normal basal transcription, but were defective in responding to GAL4-VP16, GCN4, or the dA:dT activator. Strikingly, the mutant extract responded like wild type to GAL4-HAP4. We conclude that ADA2 potentiates the activity of one class of acidic activation domain but not a second class.

Amino Acid Sequence↗
PubMed1992

Conservation and evolution of transcriptional mechanisms in eukaryotes.

Eukaryotic transcriptional activators play key roles in controlling cell growth and specifying embryonic development. These activators can stimulate promoters from distances up to tens of kilobases by a mechanism that is remarkably conserved in eukaryotes ranging from yeast to humans. Although the primary sequence of certain activators has also been conserved in widely divergent organisms, the regulatory roles that these factors play have been altered over evolution to fit the specific needs of the host.

Amino Acid Sequence↗
PubMed1992

Increased dosage of a transcriptional activator gene enhances iron-limited growth of Saccharomyces cerevisiae.

We have selected for genes that, when present in multiple copies, enhance growth of wild-type cells of Saccharomyces cerevisiae in an iron-limiting medium. A gene designated FUP1, for 'ferric utilization proficient', was isolated by this approach. Increased dosage of FUP1 reduces the concentration of iron in the medium required for efficient growth and confers elevated levels of iron uptake activity in iron-limited cells. Disruption of the FUP1 locus reduces wild-type iron uptake rates by 2-fold in cells grown on raffinose medium but has no effect on glucose-grown cells. DNA sequencing showed that FUP1 encodes a hydrophilic 43 kDa protein identical to MSN1, a gene encoding a transcriptional activator implicated in carbon source regulation. Our results suggest that FUP1/MSN1 also regulates synthesis of gene products involved in iron uptake.

Amino Acid Sequence↗
PubMed1992

HAP1 positive control mutants specific for one of two binding sites.

The expression of the yeast CYC1 and CYC7 genes is controlled by the HAP1 activator. A GAL4-like zinc finger (residues 1-148) specifies binding to the dissimilar sites UAS1 (of CYC1) and CYC7, and an acidic domain (residues 1307-1483) is essential for activation of transcription. To analyze how HAP1 binds to UAS1 and CYC7, we performed saturation mutagenesis of the DNA-binding domain and recovered mutants with altered activity. Class 1 mutants had a reduced activity at both UAS1 and CYC7, and class 2 mutants selectively eliminated activity at CYC7. Surprisingly, several mutants of both classes exhibited wild-type DNA binding, indicating that they were specifically defective in activation. These positive control (PC) mutants alter residues that bracket the zinc finger. We explain these mutants in a model involving cofactor proteins that bind UAS1 and CYC7 along with HAP1. The existence of PC mutants that only affect activity at CYC7 raises the possibility that different cofactors may exist for UAS1 and CYC7.

Amino Acid Sequence↗
PubMed1991

A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast.

We constructed a comprehensive cDNA library from HeLa cell mRNA in a vector that directs expression of the cDNA in Saccharomyces cerevisiae. We used this library to clone the human counterpart of the Sa. cerevisiae CCAAT-binding transcription factor, Hap2, by functional complementation of a hap2 mutation. The cDNA encoding the human Hap2 homolog encodes a protein of 257 amino acids that has a 62-amino acid carboxyl-terminal region 73% identical to the essential core region of Hap2. The amino terminus of the protein is highly enriched in glutamine residues, reminiscent of transcriptional activation domains of several other mammalian transcription factors. Analysis of human Hap2 expression reveals three major transcripts: a 4.1-kilobase species found in all cell types examined, a 7.0-kilobase species specific to B lymphocytes, and a 1.6-kilobase species that is expressed preferentially in HeLa cells and that likely corresponds to our cDNA clone. Thus, the human Hap2 homolog and related factors may play both a constitutive and cell type-specific role in gene expression. The general approach of cloning by complementation should allow the isolation of many human genes for which corresponding yeast mutations exist.

Amino Acid Sequence↗
PubMed1991

Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4.

Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.

Base Sequence↗
PubMed1991

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

Amino Acid Sequence↗
PubMed1990

Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Eukaryotic promoters contain binding sites for basic transcription factors and gene-specific activator proteins. The transcription factors interact at the TATA box, which lies close to the position of transcription initiation. Activators typically bind to distant sites that can lie kilobases away from the initiation site. The factor TFIID binds specifically to the TATA box to initiate an ordered pathway of assembly of the basic transcription factors. Biochemical analyses have shown that human and Saccharomyces cerevisiae TFIID are functionally interchangeable in vitro. To study further the functional conservation of this critical factor, we are surveying proteins from divergent organisms that can substitute in vivo for the S. cerevisiae TFIID. We report here the isolation of a unique gene from Schizosaccharomyces pombe that fully complements a null mutation in SPT15, the gene that encodes TFIID in S. cerevisiae. The Schiz. pombe gene encodes a protein 93% identical (166/178) to S. cerevisiae TFIID in a region consisting of a direct repeat.

Amino Acid Sequence↗
PubMed1990

Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors.

The interaction between the chimeric activator GAL4-VP16, consisting of the DNA binding domain of GAL4 and the acidic activation domain of VP16, and its target in the transcriptional machinery was studied in vitro. GAL4-VP16 stimulated transcription from a promoter bearing GAL4 sites, and greatly inhibited transcription from a promoter bearing binding sites for the dA:dT activator and from a basal promoter bearing only a TATA box. Mutations in the acidic domain that reduced activation from the GAL4 site promoter also reduced inhibition from the dA:dT promoter, indicating a similar interaction between VP16 and its target in both processes. Strikingly, if the DNA binding domain of GAL4-VP16 was occupied by a GAL4 site oligonucleotide, the protein inhibited activation by the dA:dT activator but did not inhibit basal transcription. We propose that, under these conditions, GAL4-VP16 acted to titrate an "adaptor" that bridges an interaction between the upstream activator and the basic transcriptional machinery at the TATA box.

Binding Sites↗
PubMed1990

Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements.

In this report we study the effects of internal deletions of the yeast transcriptional activator HAP1 (CYP1) on activity at two dissimilar DNA binding sites, upstream activation sequence 1 (UAS1) of CYC1 (iso-1-cytochrome c) and CYC7 (iso-2-cytochrome c). These deletions remove up to 1061 amino acids of the 1483-residue protein and bring the carboxyl-terminal acidic activation domain closer to the amino-terminal DNA-binding domain. Surprisingly, the deletions have opposite effects at the two sites; activity at UAS1 increases with deletion size, while activity at CYC7 decreases. The mutant with the largest deletion, mini-HAP1, has no measurable activity at CYC7 but binds normally to the site in vitro. In contrast, a protein with the DNA-binding domain of HAP1 fused to the acidic activation domain of GAL4 is active at both UAS1 and CYC7. These findings are discussed in the context of two models that suggest how the DNA sequence can alter the activity of the bound HAP1. In a separate experiment, we generate a mutation in the DNA-binding domain of HAP1 that requires the addition of zinc for binding to either UAS1 or CYC7 in vitro. This finding shows that a zinc finger anchors DNA binding to both types of HAP1 sites.

Chromosome Deletion↗
PubMed1990

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.

Amino Acids↗
PubMed1989

Mutations that alter transcriptional activation but not DNA binding in the zinc finger of yeast activator HAPI.

Transcription of eukaryotic genes requires an interaction between transcription factors that bind to the TATA box region, and transcriptional activators that bind to upstream activating sequences (UASs) or enhancers. Several yeast upstream transcriptional activators, such as GCN4, GAL4 and HAP1, seem to contain separate domains for binding to DNA and activating transcription. The expression of the cytochrome genes CYC1 and CYC7 is controlled by HAP1, which binds to dissimilar DNA sequences in UAS1 of CYC1 and the UAS of CYC7. HAP1 has a zinc-finger DNA-binding domain between amino-acid residues 1 and 148, and a highly acidic C-terminal activation domain between residues 1,308 and 1,483 (ref. 10). A mutant allele of the HAP1 gene, HAP1-18, leads to a change in Ser 63 to Arg 63, immediately adjacent to the zinc finger in the DNA-binding domain. The HAP1-18 mutation specifically abolishes the ability of the protein to bind to UAS1, but greatly increases the ability of the protein to activate transcription of CYC7. We now report that this increase in activation is mediated solely by the CYC7 UAS and the HAP1-18 protein, and also, that it is not caused by an altered binding affinity of the protein for the CYC7 UAS. Furthermore, even by substituting other amino acids at position 63 and over-expressing the resulting derivatives in vivo we were unable to increase activity at the UAS of CYC7 to the level obtained with HAP1-18. This rules out the possibility that the HAP1-18 mutation increases transcriptional activation by abolishing competition by UAS1 and UAS1-like sites for the protein. We thus conclude that HAP1-18 is a better activator of transcription than the wild-type protein when bound to the UAS of CYC7. Moreover, our findings indicate that in addition to the acidic activation domain, the zinc-finger DNA-binding domain participates directly in the activation of transcription.

Binding Sites↗
PubMed1989

N-terminal deletions of a mitochondrial signal sequence in yeast. Targeting information of delta-aminolevulinate synthase is encoded in non-overlapping regions.

A series of N-terminal deletions of the mitochondrial targeting sequence of the HEM1 product, delta-aminolevulinate synthase, was constructed. Targeting ability of mutant signals was assayed in vivo by fusion to beta-galactosidase or to the mature delta-aminolevulinate synthase. In the former case, the subcellular location of the fusion proteins provided a measure of import efficiency. In the later case, constructs were tested for complementation of delta hem 1 strains. We found this complementation assay to be particularly sensitive if the delta hem 1 strain is rho-. The results of these experiments, together with experiments deleting the signal from the carboxyl end, indicate that the targeting information is encoded in non-overlapping regions of the HEM1 signal.

5-Aminolevulinate Synthetase↗
PubMed1989

Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT15 suppressor of Ty element insertions.

We report the cloning of the gene that encodes the yeast TATA binding protein TFIID. TFIID contains 240 amino acids and has no obvious sequence similarity to other known proteins. TFIID was synthesized in vitro and in two separate assays behaved identically to the protein purified from yeast. TFIID bound to TATA elements from the adenovirus major late promoter (TATAAAA) and the yeast LEU2 promoter (TATTTAA) and formed protein-DNA complexes stable to electrophoresis only in the presence of TFIIA. In vitro-synthesized yeast TFIID also complemented a mammalian in vitro transcription system that lacked TFIID. Comparison of the yeast TFIID gene with the yeast SPT15 gene (suppressor of Ty element insertions) showed that the two genes are identical. This finding indicates that the yeast TFIID activity defined in vitro is responsible for specific transcription in vivo.

Amino Acid Sequence↗
PubMed1989

Five intermediate complexes in transcription initiation by RNA polymerase II.

A native gel electrophoresis DNA binding assay was used to resolve complexes formed on the adenovirus Major Late Promoter by general transcription factors and RNA polymerase II. Five sets of complexes containing distinct components were identified. These complexes were generated by sequential binding of TFIID, TFIIA, TFIIB, RNA polymerase II, and TFIIE. The relative positions of each of the factors in the complexes were determined by DNAase I footprint analysis. TFIIA, derived from yeast or mammalian cells, formed a complex with yeast TFIID and the TATA element. TFIIB bound to this complex and probably acts as a "bridge" to the polymerase and the initiation site. The addition of ATP or dATP, necessary for "activation" of transcription, resulted in an alteration of the footprint in the +20 to +30 region, the same area protected upon addition of TFIIE to the initiation complex. Addition of ribonucleotide triphosphates generated new complexes that contained accurately initiated transcripts associated with the transcription machinery and the template DNA. A model for the interactions of components in initiation of transcription by RNA polymerase II is proposed.

Adenoviridae↗
PubMed1989

Functional dissection and sequence of yeast HAP1 activator.

We present the DNA sequence and a functional dissection of the 1483 residue yeast activator HAP1. Salient results are, first, a single DNA binding domain (1-148) specifies binding to the two target sites of different sequence, UAS1 and CYC7. This domain contains a cysteine-rich zinc finger, and mutation of either of two cysteines abolishes binding to both sites. Second, mutations that specifically abolish binding to UAS1 or to CYC7 exist. These changes lie either in the residue immediately amino-terminal to the finger or in sequences carboxyl to the finger. Thus, both the base of the finger and carboxyl flanking residues are involved in specific DNA binding. Third, a distinct region (residues 245-445) mediates heme induction by masking the DNA binding domain in the absence of inducer; heme counteracts this masking, perhaps by interacting with a repeat sequence of metal binding character in this region. While sequences between 445 and 1308 have no obvious function, a highly acidic carboxyl terminus mediates transcriptional activation by HAP1.

Amino Acid Sequence↗