Yeast Genes Research Articles

Metabolic energy variation of yeast affects its antioxidant properties in beer brewing

Oxidation destroys the flavor and freshness of the beer, and yeast with improved antioxidant activity would benefit the flavor stability of the beer. The aim of this work was to study the antioxidant networks of lager yeast for brewing industry. Through transcriptome and metabolome analysis, we were able to map changes in anti-oxidant pathways and transcription of genes in lager yeast. Results suggested that metabolic energy variation in yeast might be the main reason for the improved anti-oxidant capacity. Lower metabolic rate kept the yeast at relatively lower respiratory status at the end of fermentation, thereby prolonging the lifespan and potentially supporting its usage in serial fermentations. Up-regulation of mannose synthesis and hexose transport strengthened the cell structure, which may have contributed to the anti-oxidant capacity of yeast. A deeper understanding of the globally regulation pattern of anti-oxidation in lager yeast is expected to support the design of strain development strategies for improved anti-oxidant capacity of yeast for fermentation industry.

Quantifying absolute gene expression profiles reveals distinct regulation of central carbon metabolism genes in yeast.

In addition to controlled expression of genes by specific regulatory circuits, the abundance of proteins and transcripts can also be influenced by physiological states of the cell such as growth rate and metabolism. Here we examine the control of gene expression by growth rate and metabolism, by analyzing a multi-omics dataset consisting of absolute-quantitative abundances of the transcriptome, proteome, and amino acids in 22 steady-state yeast cultures. We find that transcription and translation are coordinately controlled by the cell growth rate via RNA polymerase II and ribosome abundance, but they are independently controlled by nitrogen metabolism via amino acid and nucleotide availabilities. Genes in central carbon metabolism, however, are distinctly regulated and do not respond to the cell growth rate or nitrogen metabolism as all other genes. Understanding these effects allows the confounding factors of growth rate and metabolism to be accounted for in gene expression profiling studies.

Complete biosynthesis of the potential medicine icaritin by engineered Saccharomyces cerevisiae and Escherichia coli

Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase III clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum (EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4’-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria (higher pH than cytoplasm) of the 8-prenylkaempferol–producing yeast strain or co-culturing the 8-prenylkaempferol–producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.

The yeast ISW1b ATP-dependent chromatin remodeler is critical for nucleosome spacing and dinucleosome resolution

Isw1 and Chd1 are ATP-dependent nucleosome-spacing enzymes required to establish regular arrays of phased nucleosomes near transcription start sites of yeast genes. Cells lacking both Isw1 and Chd1 have extremely disrupted chromatin, with weak phasing, irregular spacing and a propensity to form close-packed dinucleosomes. The Isw1 ATPase subunit occurs in two different remodeling complexes: ISW1a (composed of Isw1 and Ioc3) and ISW1b (composed of Isw1, Ioc2 and Ioc4). The Ioc4 subunit of ISW1b binds preferentially to the H3-K36me3 mark. Here we show that ISW1b is primarily responsible for setting nucleosome spacing and resolving close-packed dinucleosomes, whereas ISW1a plays only a minor role. ISW1b and Chd1 make additive contributions to dinucleosome resolution, such that neither enzyme is capable of resolving all dinucleosomes on its own. Loss of the Set2 H3-K36 methyltransferase partly phenocopies loss of Ioc4, resulting in increased dinucleosome levels with only a weak effect on nucleosome spacing, suggesting that Set2-mediated H3-K36 trimethylation contributes to ISW1b-mediated dinucleosome separation. The H4 tail domain is required for normal nucleosome spacing but not for dinucleosome resolution. We conclude that the nucleosome spacing and dinucleosome resolving activities of ISW1b and Chd1 are critical for normal global chromatin organisation.

Expression attenuation as a mechanism of robustness against gene duplication

Gene duplication is ubiquitous and a major driver of phenotypic diversity across the tree of life, but its immediate consequences are not fully understood. Deleterious effects would decrease the probability of retention of duplicates and prevent their contribution to long-term evolution. One possible detrimental effect of duplication is the perturbation of the stoichiometry of protein complexes. Here, we measured the fitness effects of the duplication of 899 essential genes in the budding yeast using high-resolution competition assays. At least 10% of genes caused a fitness disadvantage when duplicated. Intriguingly, the duplication of most protein complex subunits had small to nondetectable effects on fitness, with few exceptions. We selected four complexes with subunits that had an impact on fitness when duplicated and measured the impact of individual gene duplications on their protein-protein interactions. We found that very few duplications affect both fitness and interactions. Furthermore, large complexes such as the 26S proteasome are protected from gene duplication by attenuation of protein abundance. Regulatory mechanisms that maintain the stoichiometric balance of protein complexes may protect from the immediate effects of gene duplication. Our results show that a better understanding of protein regulation and assembly in complexes is required for the refinement of current models of gene duplication.

Uncovering de novo gene birth in yeast using deep transcriptomics

De novo gene origination has been recently established as an important mechanism for the formation of new genes. In organisms with a large genome, intergenic and intronic regions provide plenty of raw material for new transcriptional events to occur, but little is know about how de novo transcripts originate in more densely-packed genomes. Here, we identify 213 de novo originated transcripts in Saccharomyces cerevisiae using deep transcriptomics and genomic synteny information from multiple yeast species grown in two different conditions. We find that about half of the de novo transcripts are expressed from regions which already harbor other genes in the opposite orientation; these transcripts show similar expression changes in response to stress as their overlapping counterparts, and some appear to translate small proteins. Thus, a large fraction of de novo genes in yeast are likely to co-evolve with already existing genes.

Developmental delay with hypotrophy associated with homozygous functionally relevant REV3L variant

REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.

Physiological function of FKBP12, a primary target of rapamycin/FK506: a newly identified role in transcription of ribosomal protein genes in yeast.

In this review, we have summarized the information from a study on FKBP12 (FK506 binding protein 12kDa) with a view to understand its drug-free, physiological roles in transcription of ribosomal protein gene in Saccharomyces cerevisiae. FKBP12 with peptidyl-prolylisomerase (PPIase) activity is widely conserved among many eukaryotes. FKBP12 is a primary target for the two structurally related drugs, FK506 and rapamycin. FKBP12 bound with FK506 or rapamycin inhibits calcineurin and target of rapamycin complex 1 (TORC1), respectively. The molecular mechanisms of the effect of FKBP12 in the presence of these drugs have been elucidated. Conversely, the physiological role of FKBP12 has been unclear, especially in yeast. Our study revealed that the deletion of FPR1 (FK506-sensitive prolinerotamase 1 gene), which encodes yeast FKBP12, induced severe growth defect synthetically with deletion of HMO1 (high mobility group family 1). HMO1 encodes an HMGB family protein involved in transcription of ribosomal component genes. Fpr1 was shown to bind specifically to the promoters of ribosomal protein genes (RPGs) dependent on Rap1 (repressor/activator binding protein 1). Importantly, Fpr1 and Hmo1 promote the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), key regulators of RPG transcription, to certain RPG promoters independently and/or cooperatively with each other. Taken together, we conclude that Fpr1 physiologically functions as transcription factor of RPGs in S. cerevisiae. To our knowledge, this is the first study to demonstrate that FKBP12 participates in ribosome synthesis independently of drugs, and it may also provide a clue to the unidentified function of other PPIase proteins.

Fission Yeast Methylenetetrahydrofolate Reductase Ensures Mitotic and Meiotic Chromosome Segregation Fidelity.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11+, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11+ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

Analysis of Genes AT5G64110 and IAA18 as Potential Downstream Targets of ARF-19

Auxin is a crucial hormone in plants that provokes many different responses involved with physiological processes of plants including lateral root development, primary root growth, and gravitropism. In the plant Arabidopsis thaliana, the hormone enhances the expression of auxin response factor (ARF) transcription factors. This study focuses on ARF-19, which is crucial for the lateral root development in plants. This work aims to identify a direct downstream target of ARF-19 and to further understand how plant transcription factors work. An Arabidopsis gene of interest was selected and inserted into a plasmid which was amplified through transformation into E. coli. The plasmid with the promoter insert of the gene was then expressed in yeast, Saccharomyces cerevisiae. Observing the expression of the gene in yeast helps us determine if ARF-19 uses the gene’s promoter to drive its transcription. Gene expression was measured using qRT-PCR, confocal microscopy, and plate reader assays. The first gene of choice was AT5G64110, however, error in the formation of the plasmid forced the transition to analyze the IAA18 gene as a potential target. Expression quantification experiments indicate that ARF-19 does not bind to the IAA18 gene promoter. This gene is not a direct target of ARF-19 through the auxin-mediated pathway. Further characterization of the IAA18 gene can reveal more about this transcription factors role in plant growth, independent of auxin.

An HD-Zip transcription factor, VvHDZ4, in grapes (Vitis vinifera L.) confers enhanced drought tolerance in transgenic tomato

BACKGROUND: HD-Zip genes encode several large and highly conserved protein families of transcription factors that play important roles in plant development and responses to environmental stress. To date, information about the involvement of HD-Zip in grape to drought response is limited. OBJECTIVE: The production of grapes is limited by a range of biotic and abiotic stresses, which cause significant losses in yield every year as well as a reduction in fruit quality. Identification and analysis of stress related genes in grapes are very important for cultivating more robust varieties with environmental stress resistance METHODS: We isolated a homeodomain-leucine zipper gene (HD-Zip), VvHDZ4, from grape, Vitis vinifera L. “Yatomi Rose”, and characterized VvHDZ4 based on analyses of its expression patterns, subcellular localization, transcription activity, and overexpression. RESULTS: We found that VvHDZ4 was highly expressed in roots and leaves, localized to nucleus, and activated the expression of reporter genes in yeast. The overexpression (OE) of VvHDZ4 led to enhanced drought stress tolerance in Solanum lycopersicum L. “Ailsa Craig” (tomato). The drought-tolerant phenotypes of the OE lines exhibited decreases in electrolyte leakage and rate of water loss, higher photosynthetic production and net photosynthesis rate, and showed up-regulation of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), all of which are key antioxidant enzymes known to be activated during drought stress. Moreover, we observed the up-regulation of four additional drought stress indicator genes, SIDREB2A, SIAREB, SIRD29 and SIERD10, in the OE lines. VvHDZ4 improved drought tolerance in tomato, and the modes of action possibly consist of reducing membrane damage, increasing photosynthetic productivity, and modulating the expression of stress defense genes. CONCLUSIONS: This work increases our understanding of the important roles HD-Zip transcription factors in the responses of plants to the environment, especially abiotic stress.

Exploring the salt- and drought-tolerant genes of alfalfa through expression library screening strategy

Salinity and drought stress are major abiotic stresses negatively affecting the growth and productivity of alfalfa (Medicago sativa L.). Exploration of genes exhibiting superior tolerance to salinity and drought stress in alfalfa will help aid target molecular breeding and developing tolerant forages. In this study, we adopted a high-efficient yeast FOX hunting system for the identification of salinity and drought tolerant genes in alfalfa. Based on the Gateway-compatible vector system, a high-quality expression library was constructed, containing 1.3 × 107 clones with an average size of 1.44 kb. Through heterologous transformation of mixed library plasmid into salt or drought sensitive yeast mutants, monoclonal resistant strains were screened and tolerant genes were captured. Eighteen salinity-tolerance genes were obtained which were involved in several pathways, containing GRAS and zinc finger transcriptional factors, PP2A interaction module, ERVT vesicle transporter and LETM transmembrane protein. Twelve drought tolerance genes were separated, including ERF and SCL transcriptional factors, CIPK and BSK protein kinases, TGL-type ligase, cPGM and cPDL protease. The mRNA transcription levels of these tolerant genes were inducible or suppressible for response to salt or drought stress conditions following quantitative PCR detection, respectively. Furthermore, heterologous transformation of ERVT and CIPK11 can improve the salt and drought stress tolerance in Arabidopsis, which indicates the conservative function of the screening gene in yeast and Arabidopsis. Obtaining these candidate genes can provide new insights for future research with respect to plant salt and drought tolerance.

Acute stress drives global repression through two independent RNA polymerase II stalling events in Saccharomyces

SUMMARYIn multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30–50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert.

Bayesian Functional Mixed-effects Models with Grouped Smoothness for Analyzing Time-course Gene Expression Data

Objective: As a result of the development of microarray technologies, gene expression levels of thousands of genes involved in a given biological process can be measured simultaneously, and it is important to study their temporal behavior to understand their mechanisms. Since the dependence between gene expression levels over time for a given gene is often too complicated to model parametrically, sparse functional data analysis has received an increasing amount of attention for analyzing such data. Methods: We propose a new functional mixed-effects model for analyzing time-course gene expression data. Specifically, the model groups individual functions with heterogeneous smoothness. The proposed method utilizes the mixed-effects model representation of penalized splines for both the mean function and the individual functions. Given noninformative or weakly informative priors, Bayesian inference on the proposed models was developed, and Bayesian computation was implemented by using Markov chain Monte Carlo methods. Results: The performance of our new model was studied by two simulation studies and illustrated using a yeast cell cycle gene expression dataset. Simulation results suggest that our proposed methods can outperform the previously used methods in terms of the mean integrated squared error. The yeast gene expression data application suggests that the proposed model with two latent groups should be used on this dataset. Conclusion: The new Bayesian functional mixed-effects model that assumes multiple groups of functions with different smoothing parameters provides an enhanced approach to analyzing timecourse gene expression data.

640. Analytical Validation of the BioFire® Bone and Joint Infection (BJI) Panel for the Identification of Bacteria, Yeast, and Antimicrobial Resistance Genes from Synovial Fluid

BackgroundThe BioFire Bone and Joint Infection (BJI) Panel is a sample-to-answer test for the qualitative detection of nearly 40 different bacteria, yeast, and antimicrobial resistance (AMR) genes in synovial fluid (SF). The panel aims to improve on current culture-based diagnostics, particularly for detection of anaerobes (e.g. Finegoldia magna, Kingella kingae, Cutibacterium, Anaerococcus and Peptoniphilus species, and others) in about an hour. Analytical performance of the panel (Limit of Detection (LoD), analytical reactivity and specificity, interference, reproducibility), and specimen storage conditions are described.MethodsLoD for each analyte was estimated from serial dilutions and confirmed at the lowest titer with ≥95% detection. A collection of >350 isolates representing genetic and geographic diversity of analytes was tested near LoD to assess analytical reactivity, and more than 420 near-neighbor, commensal, pathogenic, or environmental off-panel species were evaluated for assay specificity. Reproducibility was evaluated in a multi-laboratory multi-variable study, and the impact of storage and potentially interfering substances on the accuracy of test results was also assessed. Testing was performed with Investigational Use Only kits.ResultsThe confirmed LoD for bacteria and yeast ranged from 100 - 10,000 CFU/mL. Sequence analysis and testing demonstrated clinically appropriate specificity and reactivity with a variety of isolates and different AMR gene types. Accurate and reproducible organism and AMR gene detection was observed with repeated testing of samples over several days (99.9% agreement with the expected results), and detection was not affected by potentially interfering substances nor by refrigerated sample storage.ConclusionThe BioFire BJI Panel is a robust, accurate, and easy-to-use multiplex PCR test capable of detecting many aerobic and anaerobic bacteria, yeast, and AMR genes in synovial fluid specimens. Rapid and reliable molecular detection of possible BJI pathogens may advance the diagnosis and effective management of bone and joint infections.NoteThis panel has not been evaluated by the FDA or other regulatory agencies for diagnostic use.DisclosuresNicholas Francis, n/a, BioFire Diagnostics (Employee) Laurence Barbier, n/a, Biomerieux (Employee) Caroline Dubost, n/a, Biomerieux (Employee) Elodie Billet, n/a, Biomerieux (Employee) Joel Manwaring, n/a, BioFire Diagnostics (Employee) Josh Southwick, n/a, BioFire Diagnostics (Employee) Tyson Dawson, n/a, BioFire Diagnostics (Employee) Jess Gann, n/a, BioFire Diagnostics (Employee) Kevin Ekins, n/a, BioFire Diagnostics (Employee) Jennifer Arce, MS, BioFire Diagnostics/BioMerieux (Employee) Briana Flaherty, n/a, BioFire Diagnostics (Employee) Harmonie Durand, n/a, Biomerieux (Employee) Chris Cantrell, n/a, Biomerieux (Employee) Elizabeth Amiott, n/a, BioFire Diagnostics (Employee)

Transcriptional Regulatory Networks Involved in C3-C4 Alcohol Stress Response and Tolerance in Yeast.

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. In this study, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3-C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3-C4 alcohols by perturbing the transcriptional regulatory network.

Flocculation Type and the Lg-FLO1 Gene of Bottom-Fermenting Yeast Are Derived from Top-Fermenting Yeast

Flocculation is important in beer fermentation because its affects both the process of fermentation and the flavor characteristics of the end product. In this study, meiotic segregant Saccharomyces cerevisiae Y7, which exhibits the NewFlo type flocculation desirable for brewing, was isolated from the top-fermenting yeast S. cerevisiae Y6. Next-generation sequencing showed that the Y7 genome contained two predicted genes (homologs of FLO5 and Lg-FLO1) that might be involved in NewFlo-type flocculation. Two transformants were constructed by inserting a drug resistance gene (kanMX6) into the respective genes. Flocculation was maintained when kanMX6 was inserted into the FLO5 homolog, but significantly reduced in the transformant when kanMX6 was inserted into one of the Lg-FLO1 loci. These results suggested that the Lg-FLO1 homolog is involved in the NewFlo-type flocculation of the top-fermenting yeast S. cerevisiae Y7. The bottom-fermenting S. pastorianus yeast strains commonly used in brewing are widely believed to derive from a natural hybrid of S. eubayanus and S. cerevisiae (i.e., a top-fermenting parent strain). Based on our results, it is speculated that the flocculation type and Lg-FLO1 gene of bottom-fermenting yeast are derived from top-fermenting yeast. These findings are expected to contribute to cross breeding work in the brewing industry and to the control of flocculation of top-fermenting yeast.

Correction: Chromatin remodeler Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes in yeast.

Correction: Chromatin remodeler Ino80C acts independently of H2A.Z to evict promoter nucleosomes and stimulate transcription of highly expressed genes in yeast.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

A Rab escort protein regulates the MAPK pathway that controls filamentous growth in yeast

MAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.