Budding Yeast Saccharomyces Cerevisiae Research Articles

Rapid identification of reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae

Infertility affects ∼12 % of couples, with environmental chemical exposure as a potential contributor. Of the chemicals that are actively manufactured, very few are assessed for reproductive health effects. Rodents are commonly used to evaluate reproductive effects, which is both costly and time consuming. Thus, there is a pressing need for rapid methods to test a broader range of chemicals. Here, we developed a strategy to evaluate large numbers of chemicals for reproductive toxicity via a yeast, S. cerevisiae high-throughput assay to assess gametogenesis as a potential new approach method (NAM). By simultaneously assessing chemicals for growth effects, we can distinguish if a chemical affects gametogenesis only, proliferative growth only or both. We identified a well-known mammalian reproductive toxicant, bisphenol A (BPA) and ranked 19 BPA analogs for reproductive harm. By testing mixtures of BPA and its analogs, we found that BPE and 17 β-estradiol each together with BPA showed synergistic effects that worsened reproductive outcome. We examined an additional 179 environmental chemicals including phthalates, pesticides, quaternary ammonium compounds and per- and polyfluoroalkyl substances and found 57 with reproductive effects. Many of the chemicals were found to be strong reproductive toxicants that have yet to be tested in mammals. Chemicals having affect before meiosis I division vs. meiosis II division were identified for 16 gametogenesis-specific chemicals. Finally, we demonstrate that in general yeast reproductive toxicity correlates well with published reproductive toxicity in mammals illustrating the promise of this NAM to quickly assess chemicals to prioritize the evaluation for human reproductive harm.

Delayed luminescence to monitor growth stages and assess the entropy of Saccharomyces cerevisiae

Delayed luminescence (DL) refers to the photon-induced ultra-weak luminescence emitted by samples after the light source is switched off. As a noninvasive method for health monitoring and disease diagnosis, DL has attracted increasing attention. The further development of this technology is valuable for the study of complex biological processes, such as different growth stages. If such studies were to be conducted in humans, large numbers of subjects of all ages would need to be recruited, and individual differences would be inevitable. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has a short population lifespan, and the growth phases can be monitored within dozens of hours. Therefore, S. cerevisiae is an ideal model organism for research. In this study, we investigated the physiological characteristics and DL emission of S. cerevisiae during growth in glucose-based media and entry into stationary phase, and the results showed that DL kinetic curves of yeast cells in the growing phase were obviously separated from those of stationary phase cells. Moreover, the metabolic and physiological characteristics of the yeast cell population were discussed using the DL emission parameters I0, τ and γ. We also discussed the possibility of assessing entropy using DL emission parameters. Our research demonstrates the potential of this technology to be used in wider applications.

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

Identification of distinct active pools of yeast serine palmitoyltransferase in sub-compartments of the ER.

Sphingolipids (SPs) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by the serine palmitoyltransferase (SPT). In budding yeast (Saccharomyces cerevisiae), SPT is negatively regulated by the two proteins, Orm1 and Orm2. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane or the lysosome (or vacuole in yeast). Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, measuring lipid metabolic enzyme activity in different cellular sub-compartments has been challenging. Combining a nanobody recruitment approach with SP flux analysis, we show that the nuclear endoplasmic reticulum (ER)-localized SPT and the peripheral ER localized SPT pools are differentially active. Thus, our data add another layer to the complex network of SPT regulation. Moreover, combining lipid metabolic enzyme re-localization with flux analysis serves as versatile tool to measure lipid metabolism with subcellular resolution.

Measuring Transcription Dynamics of Individual Genes Inside Living Cells.

Transcription is a highly dynamic process, which, for many genes, occurs in stochastic bursts. Studying what regulates these stochastic bursts requires visualization and quantification of transcription dynamics in single living cells. Such measurements of bursting can be accomplished by labeling nascent transcripts of single genes fluorescently with the MS2 and PP7 RNA labeling techniques. Live-cell single-molecule microscopy of transcription in real time allows for the extraction of transcriptional bursting kinetics inside single cells. This chapter describes how to set up the MS2 or PP7 RNA labeling system of endogenous genes in both budding yeast (Saccharomyces cerevisiae) and mammalian cells (mouse embryonic stem cells). We include how to genetically engineer the cells with the MS2 and PP7 system, describe how to perform the live-microscopy experiments and discuss how to extract transcriptional bursting parameters of the genes of interest.

Ecological inducers of the yeast filamentous growth pathway reveal environment-dependent roles for pathway components.

Signaling modules, such as mitogen-activated protein kinase (MAPK) pathways, are evolutionarily conserved drivers of cell differentiation and stress responses. In many fungal species including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model budding yeast Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was used, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found that induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Combinations of inducers (galactose and ethanol) stimulated the pathway to near-maximal levels, which showed dispensability of several fMAPK pathway components (e.g., mucin sensor, p21-activated kinase), but not others (e.g., adaptor, MAPKKK) and required the Ras2-protein kinase A pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments. IMPORTANCE Filamentous growth is a cell differentiation response and important aspect of fungal biology. In plant and animal fungal pathogens, filamentous growth contributes to virulence. One signaling pathway that regulates filamentous growth is an evolutionarily conserved MAPK pathway. The yeast Saccharomyces cerevisiae is a convenient model to study MAPK-dependent regulation of filamentous growth, although the inducers of the pathway are not clear. Here, we exposed yeast cells to ecologically relevant compounds (e.g., plant compounds), which identified new inducers of the MAPK pathway. In combination, the inducers activated the pathway to near-maximal levels but did not cause detrimental phenotypes associated with previously identified hyperactive alleles. This context allowed us to identify conditional bypass for multiple pathway components. Thus, near-maximal induction of a MAPK pathway by ecologically relevant inducers provides a powerful tool to assess cellular signaling during a fungal differentiation response.

A3DyDB: exploring structural aggregation propensities in the yeast proteome

BackgroundThe budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a well-established model system for studying protein aggregation due to the conservation of essential cellular structures and pathways found across eukaryotes. However, limited structural knowledge of its proteome has prevented a deeper understanding of yeast functionalities, interactions, and aggregation.ResultsIn this study, we introduce the A3D yeast database (A3DyDB), which offers an extensive catalog of aggregation propensity predictions for the S. cerevisiae proteome. We used Aggrescan 3D (A3D) and the newly released protein models from AlphaFold2 (AF2) to compute the structure-based aggregation predictions for 6039 yeast proteins. The A3D algorithm exploits the information from 3D protein structures to calculate their intrinsic aggregation propensities. To facilitate simple and intuitive data analysis, A3DyDB provides a user-friendly interface for querying, browsing, and visualizing information on aggregation predictions from yeast protein structures. The A3DyDB also allows for the evaluation of the influence of natural or engineered mutations on protein stability and solubility. The A3DyDB is freely available at http://biocomp.chem.uw.edu.pl/A3D2/yeast.ConclusionThe A3DyDB addresses a gap in yeast resources by facilitating the exploration of correlations between structural aggregation propensity and diverse protein properties at the proteome level. We anticipate that this comprehensive database will become a standard tool in the modeling of protein aggregation and its implications in budding yeast.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Spindle Position Checkpoint Kinase Kin4 Regulates Organelle Transport in Saccharomyces cerevisiae.

Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells.

Uncovering diffusive states of the yeast membrane protein, Pma1, and how labeling method can change diffusive behavior.

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2. The track diffusivity distributions of these two populations of single-particle tracks differ significantly, demonstrating that labeling method can be an important determinant of diffusive behavior. We also applied perturbation expectation maximization (pEMv2) (Koo and Mochrie in Phys Rev E 94(5):052412, 2016), which sorts trajectories into the statistically optimum number of diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the tracks into two diffusive states: an essentially immobile state and a more mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much smaller ([Formula: see text]) than the mobile fraction of TRAP-labeled Pma1 tracks ([Formula: see text]). In addition, the diffusivity of Pma1-mEos3.2's mobile state is several times smaller than the diffusivity of TRAP-labeled Pma1's mobile state. Thus, the two different labeling methods give rise to very different overall diffusive behaviors. To critically assess pEMv2's performance, we compare the diffusivity and covariance distributions of the experimental pEMv2-sorted populations to corresponding theoretical distributions, assuming that Pma1 displacements realize a Gaussian random process. The experiment-theory comparisons for both the TRAP-labeled Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach.

The Fission Yeast Mating-Type Switching Motto: "One-for-Two" and "Two-for-One".

Schizosaccharomyces pombe is an ascomycete fungus that divides by medial fission; it is thus commonly referred to as fission yeast, as opposed to the distantly related budding yeast Saccharomyces cerevisiae. The reproductive lifestyle of S. pombe relies on an efficient genetic sex determination system generating a 1:1 sex ratio and using alternating haploid/diploid phases in response to environmental conditions. In this review, we address how one haploid cell manages to generate two sister cells with opposite mating types, a prerequisite to conjugation and meiosis. This mating-type switching process depends on two highly efficient consecutive asymmetric cell divisions that rely on DNA replication, repair, and recombination as well as the structure and components of heterochromatin. We pay special attention to the intimate interplay between the genetic and epigenetic partners involved in this process to underscore the importance of basic research and its profound implication for a better understanding of chromatin biology.

Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9.

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiaegenome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressingguide RNAs(gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Caught in the act-Visualizing ribonucleases during eukaryotic ribosome assembly.

Ribosomes are essential macromolecular machines responsible for translating the genetic information encoded in mRNAs into proteins. Ribosomes are composed of ribosomal RNAs and proteins (rRNAs and RPs) and the rRNAs fulfill both catalytic and architectural functions. Excision of the mature eukaryotic rRNAs from their precursor transcript is achieved through a complex series of endoribonucleolytic cleavages and exoribonucleolytic processing steps that are precisely coordinated with other aspects of ribosome assembly. Many ribonucleases involved in pre-rRNA processing have been identified and pre-rRNA processing pathways are relatively well defined. However, momentous advances in cryo-electron microscopy have recently enabled structural snapshots of various pre-ribosomal particles from budding yeast (Saccharomyces cerevisiae) and human cells to be captured and, excitingly, these structures not only allow pre-rRNAs to be observed before and after cleavage events, but also enable ribonucleases to be visualized on their target RNAs. These structural views of pre-rRNA processing in action allow a new layer of understanding of rRNA maturation and how it is coordinated with other aspects of ribosome assembly. They illuminate mechanisms of target recognition by the diverse ribonucleases involved and reveal how the cleavage/processing activities of these enzymes are regulated. In this review, we discuss the new insights into pre-rRNA processing gained by structural analyses and the growing understanding of the mechanisms of ribonuclease regulation. This article is categorized under: Translation > Ribosome Biogenesis RNA Processing > rRNA Processing.

Sequence features around cleavage sites are highly conserved among different species and a critical determinant for RNA cleavage position across eukaryotes

RNA degradation is one of the critical steps for control of gene expression, and endonucleolytic cleavage-dependent RNA degradation is conserved among eukaryotes. Some cleavage sites are secondarily capped in the cytoplasm and identified using the Cap analysis of gene expression (CAGE) method. Although uncapped cleavage sites are widespread in eukaryotes, comparatively little information has been obtained about these sites using CAGE-based degradome analysis. Previously, we developed the truncated RNA-end sequencing (TREseq) method in plant species and used it to acquire comprehensive information about uncapped cleavage sites; we observed G-rich sequences near cleavage sites. However, it remains unclear whether this finding is general to other eukaryotes. In this study, we conducted TREseq analyses in fruit flies (Drosophila melanogaster) and budding yeast (Saccharomyces cerevisiae). The results revealed specific sequence features related to RNA cleavage in D.melanogaster and S.cerevisiae that were similar to sequence patterns in Arabidopsis thaliana. Although previous studies suggest that ribosome movements are important for determining cleavage position, feature selection using a random forest classifier showed that sequences around cleavage sites were major determinant for cleaved or uncleaved sites. Together, our results suggest that sequence features around cleavage sites are critical for determining cleavage position, and that sequence-specific endonucleolytic cleavage-dependent RNA degradation is highly conserved across eukaryotes.

Growth Rate Evaluation of the Budding Yeast Saccharomyces cerevisiae Cells Carrying Endogenously Expressed Fluorescent Protein Fusions.

Fluorescent proteins within fluorescent fusions have been reported to affect cellular growth fitness via altering native protein function and intracellular localization. Here we report in detail a procedure to analyze the growth characteristics of yeast cells expressing such fusions in comparison to unmodified parental strain. This approach can serve as an initial step in fluorescent protein characterization in vivo.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice.

The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development.

Comparative Study of Quantitative TSS Maps among Yeast Strains Demonstrates Key Genetic Sites Required for TSS Selection

Gene transcription is finely regulated to ensure proper cellular functions. Transcription initiation is a critical step of gene regulation as it integrates most regulatory signals, determines the location of transcription start sites (TSS), and establishes the transcript abundance. Recent studies have shown that transcription of most genes in eukaryotes can be initiated from multiple spatially located TSSs. Furthermore, different TSSs were used in various cell types as well as in response to environmental stress. How TSSs are selected during transcription, however, remains largely unknown. We aimed to infer important genetic variations associated with divergence of TSSs to better understand the role of genomic context in TSS selection. Specifically, we used no‐amplification non‐tagging cap analysis of gene expression (nAnT‐iCAGE) sequencing to accurately determine TSSs on a genome‐wide scale for three different populations of budding yeast (Saccharomyces cerevisiae). Comparative studies of these quantitative TSS maps and their genomic sequences showed that transversion substitutions at several positions, such as ‐8, ‐1, and +1 position relative to the TSS, coincided with divergence of the dominant TSS in many promoters. Additionally, a range of transversion substitutions upstream of the TSS at around the ‐150 position showed divergence of the dominant TSS in many promoters as well. However, many divergent TSSs did not have genetic variations nearby, suggesting that transcription factors likely played a major role in TSS selection. This study lays a solid foundation for further functional characterization of these genetic variations, contributing to a better understanding of the genetic mechanisms underlying the regulation of transcription initiation.

Mediator dynamics during heat shock in budding yeast.

The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In budding yeast (Saccharomyces cerevisiae), Mediator is recruited by activators and associates with core promoter regions, where it facilitates preinitiation complex (PIC) assembly, only transiently before Pol II escape. Interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes this association. However, Mediator occupancy and dynamics have not been examined on a genome-wide scale in yeast grown in nonstandard conditions. Here we investigate Mediator occupancy following heat shock or CdCl2 exposure, with and without depletion of Kin28. We find that Pol II occupancy shows similar dependence on Mediator under normal and heat shock conditions. However, although Mediator association increases at many genes upon Kin28 depletion under standard growth conditions, little or no increase is observed at most genes upon heat shock, indicating a more stable association of Mediator after heat shock. Unexpectedly, Mediator remains associated upstream of the core promoter at genes repressed by heat shock or CdCl2 exposure whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. This persistent association is strongest at promoters that bind the HMGB family member Hmo1, and is reduced but not eliminated in hmo1Δ yeast. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting altered dynamics or stronger engagement with activators under these conditions.