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L Guarente

Publications and source records attributed to L Guarente.

At least 37 records · Page 2Linked to original sources
PubMed1989

Identification of a yeast protein homologous in function to the mammalian general transcription factor, TFIIA.

The yeast homolog of the mammalian RNA polymerase II general transcription factor TFIIA has been identified by complementation of a mammalian in vitro transcription system depleted for TFIIA. Like the mammalian factor, the yeast protein does not bind DNA, alters the size of the TFIID DNase I footprint at the adenovirus major late promoter, and forms specific TFIIA-TFIID-DNA complexes which are stable during electrophoresis in native acrylamide gels. The partially purified yeast factor was used to investigate its effect on the binding of TFIID to the major late promoter. Contrary to earlier models, we find that TFIIA does not significantly change the affinity or kinetics of TFIID binding, suggesting that it acts by altering the conformation of TFIID and/or by serving as a bridge between TFIID and the other general transcription factors.

Adenoviridae↗
PubMed1989

Genetic selection for genes encoding sequence-specific DNA-binding proteins.

We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins. The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins. Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins. Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA. Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein.

Animals↗
PubMed1989

Mutational analysis of a yeast transcriptional terminator.

We have isolated and mutagenized a DNA fragment from Saccharomyces cerevisiae that specifies mRNA 3' end formation for the convergently transcribed CYC1 and UTR1 genes. An in vivo plasmid supercoiling assay previously showed that this fragment is a transcriptional terminator, and "run-on" assays shown here are consistent with this interpretation. The poly(A) sites in the mRNAs formed by the fragment are the same whether the fragment resides at the native location or at a heterologous location. No single linker substitution abolishes the fragment's activity, whereas certain large, nonoverlapping deletions have strong, deleterious effects. Therefore, the yeast terminator behaves more like rho-dependent bacterial terminators than terminators of higher eukaryotes. That a number of deletions or substitutions have different effects in the two orientations suggests that the fragment contains the sequences of two, unidirectional terminator elements.

Base Sequence↗
PubMed1989

Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.

The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with high affinity to several TATA elements that do not match the consensus TATA sequences (TATAAA and TATATA): the yeast LEU2 TATA (TATTATTTA), the simian virus 40 TATA (CTTATTTAT), and the yeast CYC1 -10 TATA (TTATACATT) all bound TFIID. Furthermore, TFIID was active in promoting transcription in vitro from the nonconsensus TATA elements. Thus, contrary to previous suggestions, the existence of nonconsensus TATA elements does not itself indicate the existence of multiple TATA-binding factors.

Base Sequence↗
PubMed1989

Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer.

The CYC1 gene of Saccharomyces cerevisiae is positively regulated by the HAP2 and HAP3 proteins, which form a heteromeric complex that binds to a CCAAT box in the upstream activation site, UAS2, and which activate transcription in a nonfermentable carbon source. We carried out a genetic analysis to identify additional trans-acting regulatory factors exerting their effects through UAS2. We present the identification and characterization of a new locus, HAP4, which is shown to encode a subunit of the DNA-binding complex at UAS2. In the hap4 mutant, the binding of HAP2 and HAP3 (HAP2/3) is not observed in vitro. The HAP4 gene is regulated transcriptionally by a carbon source, suggesting that it encodes a regulatory subunit of the bound complex. The sequence of HAP4 shows a highly acidic region, which innactivated the protein when deleted. Replacement of this region with the activation domain of GAL4 restored activity, suggesting that it provides the principal activation domain to the bound HAP2/3/4 complex.

Amino Acid Sequence↗
PubMed1988

Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases.

The yeast, Saccharomyces cerevisiae, contains two citrate synthase isoenzymes, mitochondrial (CS1) and cytosolic (CS2). In this study, we have examined the metabolic consequences of the absence of CS1, CS2, and both isoenzymes in the respective mutant strains CS1-, CS2-, and CS1-CS2-. No significant differences were found in the growth rates of the parental, CS1-, or CS2- strains when grown in the single carbon sources galactose, glycerol, lactate, pyruvate, or glutamate. However, in nonfermentable carbon sources, the lag period in growth of CS1- was approximately 4 times that of the parental strain and the CS2- mutant. This difference was found even in glutamate. The CS1- mutant failed to grow on acetate in either complete or minimal liquid medium. Total cellular citrate concentration in the CS1- compared to the parental strain was higher when the cells were grown in lactate or pyruvate. On these same substrates, the malate concentration was 2-fold higher in the CS1-mutant when compared to the parental or CS2- strains. The production of 14CO2 by CS1- from [1-14C]acetate was 36% and that from [2-14C]acetate was 9.2% of the amount from the parental or CS2- strains. The 14CO2 production from [1-14C]glutamate was 28% and 20% in CS1- and CS1-CS2-, respectively, compared to the parental strain. Since these results are not easily explained solely by the absence of mitochondrial citrate synthase enzyme, we also determined the activity of some other enzymes of the citric acid cycle and electron transport chain. We found decreased activity of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and aconitase, while the rest of the citric acid cycle enzymes and oxidative enzymes did not change significantly. The same changes in enzyme activities were found in two different yeast strains carrying the same citrate synthase mutations.

Acetates↗
PubMed1988

Isolation, sequence, and regulation by oxygen of the yeast HEM13 gene coding for coproporphyrinogen oxidase.

The HEM13 gene of Saccharomyces cerevisiae codes for coproporphyrinogen oxidase (EC 1.3.3.3) catalyzing the sixth enzymic step in the heme biosynthetic pathway. Its expression has been previously shown to be regulated negatively by heme and oxygen. We have isolated the HEM13 gene by functional complementation of a hem13 gene by functional complementation of a hem13 mutant and determined its nucleotide sequence. The open reading frame encodes a protein of 328 amino acids. Its calculated molecular weight (37,673), amino acid composition and amino-terminal sequence predicted from the DNA sequence are in agreement with those determined for the native enzyme (Camadro, J. M., Chambon, H., Jolles, J., and Labbe, P. (1986) Eur. J. Biochem. 156, 579-587). The 5' ends of the HEM13 transcripts were identified by nuclease S1 mapping; induction of HEM13 resulted in an equivalent increase of the level of all the transcripts. 5' deletion analysis revealed that DNA sequence located upstream of 409 nucleotides from the translational initiation codon was needed for depression under oxygen limitation. The loss of induction of coproporphyrinogen oxidase activity by anaerobiosis caused a considerable decrease of heme formation in anaerobic cells.

Amino Acid Sequence↗
PubMed1988

Function of a yeast TATA element-binding protein in a mammalian transcription system.

Saccharomyces cerevisiae contains a protein which is functionally similar to the mammalian TATA element-binding transcription factor, TFIID. The yeast factor substitutes for TFIID in a mammalian RNA polymerase II in vitro transcription system, forms a stable preinitiation complex on the Adenovirus-2 major late promoter, and binds specifically to the TATA boxes of the viral promoter and the yeast CYC1 promoter. Interestingly, the yeast factor promotes initiation at a distance from the TATA element typical of a mammalian system.

DNA-Binding Proteins↗
PubMed1988

Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex.

Transcription of the yeast C upsilon C1 gene (iso-1-cytochrome c) is regulated in part by the upstream activation site UAS2. Activity of UAS2 requires both the HAP2 and HAP3 activators, which bind to UAS2 in an interdependent manner. To distinguish whether these factors bound to UAS2 cooperatively or formed a complex in the absence of DNA, HAP2 and HAP3 were tagged by gene fusion to LexA and beta-galactosidase, respectively, and purified through four chromatographic steps. The copurification of LexA-HAP2, HAP3 beta-galactosidase, and UAS2 binding activity shows that HAP2 and HAP3 associate in the absence of DNA to form a multisubunit activation complex.

Bacterial Proteins↗
PubMed1988

A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable.

The S. cerevisiae HAP2 and HAP3 gene products have been shown to recognize CCAAT-containing transcription elements as a HAP2/HAP3 multisubunit heterologous complex, or heteromer. Recently, we have demonstrated that the human sequence-specific DNA-binding protein, CP1, also recognizes CCAAT-containing transcription elements as a heteromer. Mammalian cells contain at least three distinct CCAAT-binding proteins, all of which appear to be multisubunit complexes composed of heterologous subunits. One of these proteins, CP1, has DNA-binding properties that are virtually identical to the yeast HAP2/HAP3 complex. These two proteins bind to the same target sequences, make the same DNA contacts, and are affected in a similar manner by mutations in the CCAAT element. Most surprisingly, the subunits of CP1 and HAP2/HAP3 are functionally interchangeable. That is, the yeast/human hybrid complexes that are formed retain the ability to specifically recognize CCAAT elements.

Biological Evolution↗
PubMed1988

Transcription by RNA polymerase II induces changes of DNA topology in yeast.

We show that induction of transcription of a CYC1-lacZ fusion gene, borne on a yeast plasmid, causes an increase in negative superhelicity of approximately five turns. This increase is abolished by deletion of either essential element of the CYC1 promoter, the upstream activation site (UAS), or the TATA boxes. Several experiments indicate that the size of the increase is proportional to the size of the transcribed region. First, an internal deletion removing half of the CYC1-lacZ transcribed region results in a plasmid whose negative superhelicity on induction is intermediate between promoter-deletion plasmids and the parental plasmid. Second, plasmids bearing insertions of a fragment containing the putative CYC1 terminator into the CYC1-lacZ fusion gene have relative negative superhelicities proportional to the length of the truncated fusion transcripts generated. A plausible model explaining these observations is that local unwinding of the double helix by transcribing RNA polymerase generates positively supercoiled DNA, which is subsequently relaxed by a topoisomerase.

Chromosome Deletion↗
PubMed1988

Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site.

We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae. Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity. Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3. A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity. This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells. Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself. These regions bind distinct proteins found in crudely fractionated yeast extracts.

Base Sequence↗
PubMed1988

The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts.

Activation of the CYC1 upstream activation site, UAS2, and transcription of several other genes encoding respiratory functions requires the product of the regulatory gene HAP2. We report here the isolation and characterization of a second UAS2 regulatory gene, HAP3. Like mutations in HAP2, a mutation in HAP3 abolishes the activity of UAS2 and prevents growth on nonfermentable carbon sources. The HAP3 gene was cloned and, surprisingly, was found to encode two divergently transcribed, overlapping transcripts: a 570-base RNA and a 3-kilobase (kb) RNA. Chromosomal disruption experiments defined the critical region for HAP3 function to a 1.3-kb segment in which the two transcripts overlap. Analysis of the HAP3 DNA sequence showed that the 570-base transcript could encode a protein of 144 amino acids. Synthesis of the 144-amino-acid protein under regulatory control in vivo demonstrated that this protein is essential for activity of UAS2 as well as for growth on nonfermentable carbon sources. The largest open reading frame in the critical region of the 3-kb transcript is only 86 amino acids. Using site-directed mutagenesis, we demonstrated that the 86-amino-acid open reading frame was not involved in UAS2 activity. The possible role of this 3-kb antisense RNA in HAP3 expression or function is discussed.

Amino Acid Sequence↗
PubMed1987

Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner.

Transcriptional activation by the yeast CYC1 upstream activation site UAS2UP1 requires the products of both the HAP2 and HAP3 regulatory genes. We show here that both HAP2 and HAP3 in yeast extracts bind to UAS2UP1 and give rise to a single protein-DNA complex, termed C, in nondenaturing polyacrylamide gels. That both products are a part of complex C was shown by altering the mobility of the complex by fusing either HAP2 or HAP3 to beta-galactosidase. Further, methylation interference footprinting showed that sequences in UAS2UP1 contacted in complex C were identical to those contacted in either fusion protein complex. Binding was centered on the sequence TGATTGGT, also found in the UASs of other genes subject to activation by the HAP2-HAP3 system and homologous to the CCAAT box sequence found in higher cells. The binding of either HAP2 or HAP3 was abolished when synthesized in a strain mutant in the complementary HAP gene. Thus the binding of HAP2 and HAP3 to UAS2UP1 is interdependent. The involvement of multiple gene products in binding to a single site is discussed with reference to other systems in yeast and higher cells.

Cytochrome c Group↗
PubMed1987

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.

DNA, Fungal↗
PubMed1987

Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene.

We show that the yeast HAP1 activator locus encodes a protein that binds in vitro to the upstream activation site, UAS1, of the CYC1 gene (iso-1-cytochrome c). Binding of wild-type HAP1 and truncated HAP1 derivatives to UAS1 is evident in crudely fractionated yeast extracts using the gel electrophoresis DNA binding assay. The binding of HAP1 in vitro, like the activity of UAS1 in vivo, is stimulated by heme. HAP1 binds to region B, one of two portions of UAS1 shown to be important by genetic analysis of the site. Surprisingly, HAP1 binds to the same sequence as a second factor, RC2. Both HAP1 and RC2 bind to the same side of the helix, and make similar but not identical major and minor groove contacts that span two full turns. An additional factor that binds to the second important part of UAS1, the region A factor (RAF), is also identified. A model depicting the interplay of HAP1, RC2, and RAF in the control of UAS1 is presented.

Base Sequence↗