Instruct: Given a scientific claim, retrieve documents that support or refute the claim.
Query: Transcription rates in S. cerevisiae range between 7 and 12 kb/min.
A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.
Homothallic yeast cells undergo a specific pattern of mating-type switching initiated by an endonuclease encoded by the HO gene. HO transcription is affected by cell type (a, alpha, and a/alpha), by cell age (mother or daughter), and by the cell cycle. This paper investigates the sequences involved in HO transcription by replacing genomic DNA with copies mutated in vitro. A region between -1000 and 1400 (called URS1) is necessary for transcription in addition to a "TATA"-like region at -90. The 900 bp of DNA separating URS1 from the "TATA" box is not necessary for transcription nor for a/alpha repression and some measure of mother/daughter control, but it is necessary for correct cell cycle control.
Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
Budding yeast grown under continuous, nutrient-limited conditions exhibit robust, highly periodic cycles in the form of respiratory bursts. Microarray studies reveal that over half of the yeast genome is expressed periodically during these metabolic cycles. Genes encoding proteins having a common function exhibit similar temporal expression patterns, and genes specifying functions associated with energy and metabolism tend to be expressed with exceptionally robust periodicity. Essential cellular and metabolic events occur in synchrony with the metabolic cycle, demonstrating that key processes in a simple eukaryotic cell are compartmentalized in time.
Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions.
In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.
Histone acetylation and deacetylation in the yeast Saccharomyces cerevisiae occur by targeting acetyltransferase and deacetylase enzymes to gene promoters and, in an untargeted and global manner, by affecting most nucleosomes. Recently, new roles for histone acetylation have been uncovered, not only in transcription but also in DNA replication, repair and heterochromatin formation. Interestingly, specific acetylatable lysines can function as binding sites for regulatory factors. Moreover, histone deacetylation is not only repressive but can be required for gene activity.
Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.
The sirtuins are a phylogenetically conserved family of NAD(+) -dependent protein deacetylases that consume one molecule of NAD(+) for every deacetylated lysine side chain. Their requirement for NAD(+) potentially makes them prone to regulation by fluctuations in NAD(+) or biosynthesis intermediates, thus linking them to cellular metabolism. The Sir2 protein from Saccharomyces cerevisiae is the founding sirtuin family member and has been well characterized as a histone deacetylase that functions in transcriptional silencing of heterochromatin domains and as a pro-longevity factor for replicative life span (RLS), defined as the number of times a mother cell divides (buds) before senescing. Deleting SIR2 shortens RLS, while increased gene dosage causes extension. Furthermore, Sir2 has been implicated in mediating the beneficial effects of caloric restriction (CR) on life span, not only in yeast, but also in higher eukaryotes. While this paradigm has had its share of disagreements and debate, it has also helped rapidly drive the aging research field forward. S. cerevisiae has four additional sirtuins, Hst1, Hst2, Hst3, and Hst4. This review discusses the function of Sir2 and the Hst homologs in replicative aging and chronological aging, and also addresses how the sirtuins are regulated in response to environmental stresses such as CR.
We developed the Yeast Gene Order Browser (YGOB; http://wolfe.gen.tcd.ie/ygob) to facilitate visual comparisons and computational analysis of synteny relationships in yeasts. The data presented in YGOB, currently covering seven species, are based on sets of homologous genes that have been intensively manually curated based on both sequence similarity and genomic context (synteny). We reconciled different laboratories' lists of paralogous Saccharomyces cerevisiae gene pairs formed by genome duplication (ohnologs), and present near-exhaustive lists of the ohnolog pairs retained in S. cerevisiae (551, including 22 previously unidentified), Saccharomyces castellii (599), and Candida glabrata (404).
Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression.
Substitute Teacher Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF imm...
The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.
ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.
Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.
The rapid activation of gene expression in response to stimuli occurs largely through the regulation of RNA polymerase II-dependent transcription. In this Review, we discuss events that occur during the transcription cycle in eukaryotes that are important for the rapid and specific activation of gene expression in response to external stimuli. In addition to regulated recruitment of the transcription machinery to the promoter, it has now been shown that control steps can include chromatin remodelling and the release of paused polymerase. Recent work suggests that some components of signal transduction cascades also play an integral part in activating transcription at target genes.
Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life-indicative of a common evolutionary descent-this only applies to one RNAP-associated transcription factor-Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell.
Hundreds of different proteins regulate and implement transcription in Saccharomyces. Yet their interrelationships have not been investigated on a comprehensive scale. Here we determined the genome-wide binding locations of 200 transcription-related proteins, under normal and acute heat-shock conditions. This study distinguishes binding between distal versus proximal promoter regions as well as the 3' ends of genes for nearly all mRNA and tRNA genes. This study reveals (1) a greater diversity and specialization of regulation associated with the SAGA transcription pathway compared to the TFIID pathway, (2) new regulators enriched at tRNA genes, (3) a global co-occupancy network of >20,000 unique regulator combinations that show a high degree of regulatory interconnections among lowly expressed genes, (4) regulators of the SAGA pathway located largely distal to the core promoter and regulators of the TFIID pathway located proximally, and (5) distinct mobilization of SAGA- versus TFIID-linked regulators during acute heat shock.
The yeast transcriptional activator GAL4 binds specific sites on DNA to activate transcription of adjacent genes1–5. The distinct activating regions of GAL4 are rich in acidic residues and it has been suggested that these regions interact with another protein component of the transcriptional machinery (such as the TATA-binding protein or RNA polymerase II) while the DNA-binding region serves to position the activating region near the gene6,7,8. Here we show that various GAL4 derivatives, when expressed at high levels in yeast, inhibit transcription of certain genes lacking GAL4 binding sites, that more efficient activators inhibit more strongly and that inhibition does not depend on the DNA-binding domain. We suggest that this inhibition, which we call squelching, reflects titration of a transcription factor by the activating region of GAL4.
In the past years, simple organisms such as yeasts and worms have contributed a great deal to aging research. Studies pioneered in Saccharomyces cerevisiae were useful to elucidate a significant number of molecular mechanisms underlying cellular aging and to discover novel longevity genes. Importantly, these genes proved many times to be conserved in multicellular eukaryotes. Consequently, such discovery approaches are being extended to other yeast models, such as Schizosaccharomyces pombe, Candida albicans, Kluyveromyces lactis, and Cryptococcus neoformans. In fission yeast, researchers have found links between asymmetrical cell division and nutrient signaling pathways with aging. In this review, we discuss the state of knowledge on the mechanisms controlling both replicative and chronological aging in S pombe and the other emergent yeast models.
Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.
Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi). Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function. We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.
The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon. The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein. Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown. Here we show that TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2. The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3. Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4. TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2. Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm.
Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.