RESUMO
Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.
Assuntos
Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Quebras de DNA de Cadeia Dupla , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Exodesoxirribonuclease V/metabolismo , Ligação Proteica , Recombinases Rec A/metabolismoRESUMO
We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas/química , RNA Mensageiro/química , Transcrição Gênica , Alfa-Amanitina/química , Animais , Núcleo Celular/metabolismo , Cruzamentos Genéticos , DNA/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Microscopia Confocal , Microscopia de Fluorescência , Modelos Teóricos , Distribuição Normal , Reconhecimento Automatizado de Padrão , RNA Mensageiro/metabolismo , Especificidade da Espécie , Processos Estocásticos , Fatores de Transcrição/metabolismoRESUMO
Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.
Assuntos
Actinas/genética , Proteínas de Drosophila/genética , Troca Materno-Fetal/genética , Proteínas de Membrana/genética , Morfogênese/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Fenótipo , Gravidez , Transdução de Sinais/genética , Vinculina/genética , alfa Catenina/genéticaRESUMO
Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage λ as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófago lambda/metabolismo , Biofísica/métodos , Porinas/metabolismo , Receptores Virais/metabolismo , Anisotropia , Membrana Celular/virologia , Escherichia coli/citologia , Escherichia coli/virologia , Modelos BiológicosRESUMO
The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems.
Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Instabilidade Genômica/genética , Lisogenia/genética , Proteínas Repressoras/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Genes Virais/fisiologia , Genoma Bacteriano/fisiologia , Modelos Biológicos , Organismos Geneticamente Modificados , Proteínas Repressoras/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, P(RM). We simultaneously measured the concentration of the lambda repressor CI and the number of messenger RNAs (mRNAs) from P(RM) in individual Escherichia coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/metabolismo , Bacteriófago lambda/genética , Escherichia coli/genética , Escherichia coli/virologia , Dosagem de Genes , Lisogenia/genética , Modelos Teóricos , Probabilidade , RNA Mensageiro/biossíntese , Proteínas Repressoras/metabolismo , Análise de Célula Única , Processos Estocásticos , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2-3 d typically required for image and data analysis.
Assuntos
Escherichia coli/genética , Hibridização in Situ Fluorescente/métodos , RNA Mensageiro/análise , Algoritmos , Microscopia de Fluorescência , Sondas de Oligonucleotídeos/genéticaRESUMO
Gene activity is described by the time series of discrete, stochastic mRNA production events. This transcriptional time series shows intermittent, bursty behavior. One consequence of this temporal intricacy is that gene expression can be tuned by varying different features of the time series. Here we quantify copy-number statistics of mRNA from 20 Escherichia coli promoters using single-molecule fluorescence in situ hybridization in order to characterize the general properties of these transcriptional time series. We find that the degree of burstiness is correlated with gene expression level but is largely independent of other parameters of gene regulation. The observed behavior can be explained by the underlying variation in the duration of bursting events. Using Shannon's mutual information function, we estimate the mutual information transmitted between an outside stimulus, such as the extracellular concentration of inducer molecules, and intracellular levels of mRNA. This suggests that the outside stimulus transmits information reflected in the properties of transcriptional time series.