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[This corrects the article DOI: 10.1371/journal.pbio.2000644.].
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Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.
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Adaptação Biológica , Evolução Biológica , Farmacorresistência Fúngica/genética , Genes Fúngicos , Fenótipo , Mutação , Saccharomyces cerevisiaeRESUMO
Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.
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Candida albicans/fisiologia , Evolução Molecular , Proteínas Fúngicas/metabolismo , Modelos Genéticos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/fisiologia , Adaptação Fisiológica , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Códon , Proteínas Fúngicas/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Mutação , Estabilidade Proteica , Proteoma/genética , Proteoma/metabolismo , Ribossomos/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Seleção Genética , Estresse Fisiológico , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinaçãoRESUMO
Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes.
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Evolução Molecular , Genoma Fúngico/genética , Saccharomyces cerevisiae/genética , Adaptação Biológica/genética , Meio Ambiente , Epistasia Genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Aptidão Genética , Pleiotropia Genética , Variação Genética , Fenótipo , Transcriptoma/genéticaRESUMO
Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.
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Reparo de Erro de Pareamento de DNA , Engenharia Genética/métodos , Genoma Bacteriano , Mutação , Adenosina Trifosfatases/genética , Alelos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Oligodesoxirribonucleotídeos , TemperaturaRESUMO
Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5' and 3' cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5' cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5' sequences making up the 5' enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5' enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5' enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5' enhancer's stimulatory activity is strictly dependent on and thus follows the 3' enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5' leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.
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Fator de Iniciação 3 em Eucariotos , Fases de Leitura Aberta/genética , RNA Mensageiro , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos , Proteínas de Saccharomyces cerevisiae , Região 5'-Flanqueadora , Regiões 5' não Traduzidas , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.
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Metabolismo Energético , Proteínas de Choque Térmico HSP72/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.
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Reagentes de Ligações Cruzadas/química , Fatores de Iniciação em Eucariotos/química , Formaldeído/química , Iniciação Traducional da Cadeia Peptídica/fisiologia , Northern Blotting , Western Blotting , Extratos Celulares , Fracionamento Celular/métodos , Heparina/química , Polirribossomos/fisiologiaRESUMO
Although experimental and theoretical efforts have been applied to globally map genetic interactions, we still do not understand how gene-gene interactions arise from the operation of biomolecular networks. To bridge the gap between empirical and computational studies, we i, quantitatively measured genetic interactions between â¼185,000 metabolic gene pairs in Saccharomyces cerevisiae, ii, superposed the data on a detailed systems biology model of metabolism and iii, introduced a machine-learning method to reconcile empirical interaction data with model predictions. We systematically investigated the relative impacts of functional modularity and metabolic flux coupling on the distribution of negative and positive genetic interactions. We also provide a mechanistic explanation for the link between the degree of genetic interaction, pleiotropy and gene dispensability. Last, we show the feasibility of automated metabolic model refinement by correcting misannotations in NAD biosynthesis and confirming them by in vivo experiments.
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Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Modelos Genéticos , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Inteligência Artificial , Biologia Computacional , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5' of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5' enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.
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Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/biossíntese , Fator de Iniciação 3 em Eucariotos/fisiologia , Fases de Leitura Aberta/fisiologia , Subunidades Ribossômicas Menores de Eucariotos/fisiologia , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Região 5'-Flanqueadora , Fatores de Transcrição de Zíper de Leucina Básica , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Fator de Iniciação 3 em Eucariotos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
Three yeast strains capable of utilizing methanol as sole carbon and energy source were isolated. Two were classified as Candida boidinii, while the third belonged in the genus Pichia. From these three strains, four alcohol oxidases genes were identified and the sequences of the coding regions were determined: one from each Candida boidinii (aox0673 and aox0680) and two from Pichia sp. 159 (aoxA and aoxB). Methanol induces both alcohol oxidases in Pichia sp. 159: the levels of aoxA and aoxB mRNA reach about 100% and 300%, respectively, of that of his4 mRNA. aoxA, but not aoxB, is expressed at a low level in the presence of glucose. The newly described alcohol oxidases have proper dinucleotide binding sites and PTS1-like C-terminal tripeptides, identified as important elements for peroxisomal localization.
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Oxirredutases do Álcool/genética , Candida/enzimologia , Pichia/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Candida/classificação , Candida/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Dados de Sequência Molecular , Filogenia , Pichia/classificação , Pichia/genética , Análise de Sequência de DNARESUMO
The binding of eIF2-GTP-tRNA(i)(Met) ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo. If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed. Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control. prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection. Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S 'halfmer' in prt1-1 cells. Genetic evidence suggests that 40S-60S subunit joining is not rate-limiting in the prt1-1 mutant. Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA. Other mutations that disrupt eIF2-eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo.
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Códon de Iniciação/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação Fúngica da Expressão Gênica , Biossíntese de Proteínas , Proteínas Quinases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Genótipo , Guanosina Trifosfato/metabolismo , Cinética , Mutação/genética , Proteínas Quinases/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Initiation factor 3 (eIF3) forms a multifactor complex (MFC) with eIF1, eIF2, and eIF5 that stimulates Met-tRNA(i)(Met) binding to 40S ribosomes and promotes scanning or AUG recognition. We have previously characterized MFC subcomplexes produced in vivo from affinity-tagged eIF3 subunits lacking discrete binding domains for other MFC components. Here we asked whether these subcomplexes can bind to 40S ribosomes in vivo. We found that the N- and C-terminal domains of NIP1/eIF3c, the N- and C-terminal domains of TIF32/eIF3a, and eIF5 have critical functions in 40S binding, with eIF5 and the TIF32-CTD performing redundant functions. The TIF32-CTD interacted in vitro with helices 16-18 of domain I in 18S rRNA, and the TIF32-NTD and NIP1 interacted with 40S protein RPS0A. These results suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access to the interface side for the two eIF3 segments (NIP1-NTD and TIF32-CTD) that interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex.