RESUMO
This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.
Assuntos
Quebras de DNA de Cadeia Dupla , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Software , Meiose/genética , Genoma Fúngico , Mapeamento Cromossômico/métodos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Biologia Computacional/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismoRESUMO
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. including some of the researchers involved in the 2013 work investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.
Assuntos
RNA Polimerase II , RNA Mensageiro , Fatores de Transcrição , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade de RNA , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de Zinco , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Citoplasma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear "scanning" model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s-1. We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5'-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.
Assuntos
RNA Mensageiro , Ribossomos , Saccharomyces cerevisiae , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Guanosina Trifosfato/metabolismoRESUMO
Achieving high-gravity fermentation in the industrial production of fuel ethanol, and enhancing the fermentation efficiency of high-salt raw materials, such as waste molasses, can significantly reduce wastewater output and process costs. Therefore, the development of hyperosmotic-tolerant industrial Saccharomyces cerevisiae strains, capable of resisting high-salt stress, offers both environmental and economic benefits. Our previous study highlighted the potential of CRZ1 overexpression as a strategy to improve the yeast strain's resistance to high-salt stress, however, the underlying molecular mechanisms remain unexplored. The fermentation capabilities of the CRZ1-overexpressing strain, KCR3, and its parental strain, KF7, were evaluated under condition of 1.25 M NaCl at 35 °C. Compared to KF7, KCR3 showed an 81% increase in glucose consumption (129.25 ± 0.83 g/L) and a 105% increase in ethanol production (47.59 ± 0.93 g/L), with a yield of 0.37 g/g. Comparative transcriptomic analysis showed that under high-salt stress, KCR3 exhibited significantly upregulated expression of genes associated with ion transport, stress response, gluconeogenesis, and the utilization of alternative carbon sources, while genes related to glycolysis and the biosynthesis of ribosomes, amino acids, and fatty acids were notably downregulated compared to KF7. Crz1 likely expands its influence by regulating the expression of numerous transcription factors, thereby impacting genes involved in multiple aspects of cellular function. The study revealed the regulatory mechanism of Crz1 under high-salt stress, thereby providing guidance for the construction of salt-tolerant strains.
Assuntos
Etanol , Fermentação , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Tolerância ao Sal , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal/genética , Etanol/metabolismo , Glucose/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Perfilação da Expressão GênicaRESUMO
The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.
Assuntos
Etanol , Fermentação , Temperatura Alta , Locos de Características Quantitativas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Etanol/metabolismoRESUMO
Aminoacyl-transfer RiboNucleic Acid synthetases (ARSs) are essential enzymes that catalyze the attachment of each amino acid to their cognate tRNAs. Mitochondrial ARSs (mtARSs), which ensure protein synthesis within the mitochondria, are encoded by nuclear genes and imported into the organelle after translation in the cytosol. The extensive use of next generation sequencing (NGS) has resulted in an increasing number of variants in mtARS genes being identified and associated with mitochondrial diseases. The similarities between yeast and human mitochondrial translation machineries make yeast a good model to quickly and efficiently evaluate the effect of variants in mtARS genes. Genetic screening of patients with a clinical suspicion of mitochondrial disorders through a customized gene panel of known disease-genes, including all genes encoding mtARSs, led to the identification of missense variants in WARS2, NARS2 and RARS2. Most of them were classified as Variant of Uncertain Significance. We exploited yeast models to assess the functional consequences of the variants found in these genes encoding mitochondrial tryptophanyl-tRNA, asparaginyl-tRNA, and arginyl-tRNA synthetases, respectively. Mitochondrial phenotypes such as oxidative growth, oxygen consumption rate, Cox2 steady-state level and mitochondrial protein synthesis were analyzed in yeast strains deleted in MSW1, SLM5, and MSR1 (the yeast orthologues of WARS2, NARS2 and RARS2, respectively), and expressing the wild type or the mutant alleles. Pathogenicity was confirmed for most variants, leading to their reclassification as Likely Pathogenic. Moreover, the beneficial effects observed after asparagine and arginine supplementation in the growth medium suggest them as a potential therapeutic approach.
Assuntos
Aminoacil-tRNA Sintetases , Mitocôndrias , Doenças Mitocondriais , Saccharomyces cerevisiae , Humanos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Doenças Mitocondriais/genética , Saccharomyces cerevisiae/genética , Mitocôndrias/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação de Sentido IncorretoRESUMO
Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA/metabolismo , RNA/genética , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , RNA Antissenso/genética , RNA Antissenso/metabolismoRESUMO
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Assuntos
Adenosina Trifosfatases , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Ativação Transcricional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Domínios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
BACKGROUND: Novel protein-coding genes were considered to be born by re-organization of pre-existing genes, such as gene duplication and gene fusion. However, recent progress of genome research revealed that more protein-coding genes than expected were born de novo, that is, gene origination by accumulating mutations in non-genic DNA sequences. Nonetheless, the in-depth process (scenario) for de novo origination is not well understood. RESULTS: We have conceived bioinformatic analysis for sketching a scenario for de novo origination of protein-coding genes. For each de novo protein-coding gene, we firstly identified an edge of a given phylogenetic tree where the gene was born based on parsimony. Then, from a multiple sequence alignment of the de novo gene and its orthologous regions, we constructed ancestral DNA sequences of the gene corresponding to both end nodes of the edge. We finally revealed statistical features observed in evolution between the two ancestral sequences. In the analysis of the Saccharomyces cerevisiae lineage, we have successfully sketched a putative scenario for de novo origination of protein-coding genes. (1) In the beginning was GC-rich genome regions. (2) Neutral mutations were accumulated in the regions. (3) ORFs were extended/combined, and then (4) translation signature (Kozak consensus sequence) was recruited. Interestingly, as the scenario progresses from (2) to (4), the specificity of mutations increases. CONCLUSION: To the best of our knowledge, this is the first report outlining a scenario of de novo origination of protein-coding genes. Our bioinformatic analysis can capture events that occur during a short evolutionary time by directly observing the evolution of the ancestral sequences from non-genic to genic. This property is suitable for the analysis of fast evolving de novo genes.
Assuntos
Evolução Molecular , Fases de Leitura Aberta , Filogenia , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Biologia Computacional/métodos , Mutação , Genoma FúngicoRESUMO
BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.
Assuntos
Engenharia Metabólica , Ácido Mevalônico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica/métodos , Ácido Mevalônico/metabolismo , Alcenos/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SesquiterpenosRESUMO
Microbial secretions, such as metabolic enzymes, are often considered to be cooperative public goods as they are costly to produce but can be exploited by others. They create incentives for the evolution of non-producers, which can drive producer and population productivity declines. In response, producers can adjust production levels. Past studies suggest that while producers lower production to reduce costs and exploitation opportunities when under strong selection pressure from non-producers, they overproduce secretions when these pressures are weak. We challenge the universality of this trend with the production of a metabolic enzyme, invertase, by Saccharomyces cerevisiae, which catalyses sucrose hydrolysis into two hexose molecules. Contrary to past studies, overproducers evolve during evolutionary experiments even when under strong selection pressure from non-producers. Phenotypic and competition assays with a collection of synthetic strains - engineered to have modified metabolic attributes - identify two mechanisms for suppressing the benefits of invertase to those who exploit it. Invertase overproduction increases extracellular hexose concentrations that suppresses the metabolic efficiency of competitors, due to the rate-efficiency trade-off, and also enhances overproducers' hexose capture rate by inducing transporter expression. Thus, overproducers are maintained in the environment originally thought to not support public goods production.
Assuntos
Saccharomyces cerevisiae , beta-Frutofuranosidase , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Frutofuranosidase/metabolismo , beta-Frutofuranosidase/genética , Sacarose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Evolução Biológica , Hexoses/metabolismo , Regulação para Cima , Regulação Fúngica da Expressão GênicaRESUMO
As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by â¼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.
Assuntos
Membrana Celular , Retículo Endoplasmático , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/metabolismo , Membrana Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Canais de Cálcio/metabolismo , Lipídeos de Membrana/metabolismo , Fusão de Membrana , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Pressão OsmóticaRESUMO
We studied the effect of dimethyl sulfoxide (DMSO) on the biochemical and physiological parameters of S. cerevisiae yeast cells with varied energy metabolism and antioxidant status. The wild-type cells of varied genetic backgrounds and their isogenic mutants with impaired antioxidant defences (Δsod mutants) or response to environmental stress (ESR) (Δmsn2, Δmsn4 and double Δmsn2msn4 mutants) were used. Short-term exposure to DMSO even at a wide range of concentrations (2-20%) had little effect on the metabolic activity of the yeast cells and the stability of their cell membranes, but induced free radicals production and clearly altered their proliferative activity. Cells of the Δsod1 mutant showed greater sensitivity to DMSO in these conditions. DMSO at concentrations from 4 to 10-14% (depending on the strain and genetic background) activated the ESR programme. The effects of long-term exposure to DMSO were mainly depended on the type of energy metabolism and antioxidant system efficiency. Yeast cells with reduced antioxidant system efficiency and/or aerobic respiration were more susceptible to the toxic effects of DMSO than cells with a wild-type phenotype and respiro-fermentative or fully fermentative metabolism. These studies suggest a key role of stress response programs in both the processes of cell adaptation to small doses of this xenobiotic and the processes related to its toxicity resulting from large doses or chronic exposure to DMSO.
Assuntos
Antioxidantes , Dimetil Sulfóxido , Metabolismo Energético , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Metabolismo Energético/efeitos dos fármacos , Antioxidantes/metabolismo , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , MutaçãoRESUMO
Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.
Assuntos
Microscopia de Fluorescência , Nucleossomos , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/genética , Microscopia de Fluorescência/métodos , Proteína 1 de Modelagem do Nucleossomo/química , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Proteína 1 de Modelagem do Nucleossomo/genética , Imagem Individual de Molécula/métodos , DNA/química , DNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native-like environment in an almost unidirectional inside-out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short-chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Candida albicans/metabolismo , CinéticaRESUMO
The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.
Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , Histonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transcrição Gênica , Raios Ultravioleta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Reparo do DNA/genética , Histonas/metabolismo , Histonas/genética , Instabilidade Genômica/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Dano ao DNA/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Genoma Fúngico , Quebras de DNA de Cadeia Dupla/efeitos da radiação , 4-Nitroquinolina-1-Óxido/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos da radiaçãoRESUMO
Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation.
Assuntos
Cromatina , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/metabolismo , DNA/metabolismo , Ubiquitinação , Endopeptidases/metabolismo , DNA Fúngico/metabolismo , DNA Fúngico/genética , Enzimas Desubiquitinantes/metabolismo , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Ubiquitina TiolesteraseRESUMO
Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III2-IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII2 loops to conduct electron transfer between CIII2 and CIV.
Assuntos
Microscopia Crioeletrônica , Citocromos c , Saccharomyces cerevisiae , Salinidade , Eletricidade Estática , Saccharomyces cerevisiae/metabolismo , Transporte de Elétrons , Citocromos c/química , Citocromos c/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Cinética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Modelos Moleculares , Cardiolipinas/química , Cardiolipinas/metabolismoRESUMO
The C-terminal domain of RPB1 (CTD) orchestrates transcription by recruiting regulators to RNA Pol II upon phosphorylation. With CTD driving condensate formation on gene loci, the molecular mechanism behind how CTD-mediated recruitment of transcriptional regulators influences condensates formation remains unclear. Our study unveils that phosphorylation reversibly dissolves phase separation induced by the unphosphorylated CTD. Phosphorylated CTD, upon specific association with transcription regulators, forms distinct condensates from unphosphorylated CTD. Functional studies demonstrate CTD variants with diverse condensation properties exhibit differences in promoter binding and mRNA co-processing in cells. Notably, varying CTD lengths influence the assembly of RNA processing machinery and alternative splicing outcomes, which in turn affects cellular growth, linking the evolution of CTD variation/length with the complexity of splicing from yeast to human. These findings provide compelling evidence for a model wherein post-translational modification enables the transition of functionally specialized condensates, highlighting a co-evolution link between CTD condensation and splicing.
Assuntos
Processamento Alternativo , RNA Polimerase II , Saccharomyces cerevisiae , Transcrição Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Humanos , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-TraducionalRESUMO
Millions of years of evolution have optimized many biosynthetic pathways by use of multi-step catalysis. In addition, multi-step metabolic pathways are commonly found in and on membrane-bound organelles in eukaryotic biochemistry. The fundamental mechanisms that facilitate these reaction processes provide strategies to bioengineer metabolic pathways in synthetic chemistry. Using Brownian dynamics simulations, here we modeled intermediate substrate transportation of colocalized yeast-ester biosynthesis enzymes on the membrane. The substrate acetate ion traveled from the pocket of aldehyde dehydrogenase to its target enzyme acetyl-CoA synthetase, then the substrate acetyl CoA diffused from Acs1 to the active site of the next enzyme, alcohol-O-acetyltransferase. Arranging two enzymes with the smallest inter-enzyme distance of 60 Å had the fastest average substrate association time as compared with anchoring enzymes with larger inter-enzyme distances. When the off-target side reactions were turned on, most substrates were lost, which suggests that native localization is necessary for efficient final product synthesis. We also evaluated the effects of intermolecular interactions, local substrate concentrations, and membrane environment to bring mechanistic insights into the colocalization pathways. The computation work demonstrates that creating spatially organized multi-enzymes on membranes can be an effective strategy to increase final product synthesis in bioengineering systems.