RESUMEN
Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Genes Bacterianos , Estabilidad del ARN/genética , ARN Bacteriano/metabolismo , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Hierro/farmacología , Cinética , Modelos Genéticos , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.
Asunto(s)
Anabaena/genética , Regulación Bacteriana de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Anabaena/crecimiento & desarrollo , Anabaena/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Periodicidad , Transporte de Proteínas , Factores de Transcripción/genéticaRESUMEN
The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.
Asunto(s)
Bacteriófago lambda/genética , Cromosomas Bacterianos/ultraestructura , ADN Viral/genética , Escherichia coli/virología , Sitios de Unión , Mapeo Cromosómico , Difusión , Escherichia coli/metabolismo , Genoma Viral , Proteínas Luminiscentes/metabolismo , Lisogenia , Recombinación Genética , Proteínas Virales/genética , Integración Viral , Proteína Fluorescente RojaRESUMEN
The Technion autonomous underwater vehicle (TAUV) is an ongoing project aiming to develop and produce a small AUV to carry on research missions, including payload dropping, and to demonstrate acoustic communication. Its navigation system is based on an inertial navigation system (INS) aided by a Doppler velocity log (DVL), magnetometer, and pressure sensor (PS). In many INSs, such as the one used in TAUV, only the velocity vector (provided by the DVL) can be used for aiding the INS, i.e., enabling only a loosely coupled integration approach. In cases of partial DVL measurements, such as failure to maintain bottom lock, the DVL cannot estimate the vehicle velocity. Thus, in partial DVL situations no velocity data can be integrated into the TAUV INS, and as a result its navigation solution will drift in time. To circumvent that problem, we propose a DVL-based vehicle velocity solution using the measured partial raw data of the DVL and additional information, thereby deriving an extended loosely coupled (ELC) approach. The implementation of the ELC approach requires only software modification. In addition, we present the TAUV six degrees of freedom (6DOF) simulation that includes all functional subsystems. Using this simulation, the proposed approach is evaluated and the benefit of using it is shown.
RESUMEN
Cell-to-cell variations in protein abundance, called noise, give rise to phenotypic variability between isogenic cells. Studies of noise have focused on stochasticity introduced at transcription, yet the effects of post-transcriptional regulatory processes on noise remain unknown. We study the effects of RyhB, a small-RNA of Escherichia coli produced on iron stress, on the phenotypic variability of two of its downregulated target proteins, using dual chromosomal fusions to fluorescent reporters and measurements in live individual cells. The total noise of each of the target proteins is remarkably constant over a wide range of RyhB production rates despite cells being in stress. In fact, coordinate downregulation of the two target proteins by RyhB reduces the correlation between their levels. Hence, an increase in phenotypic variability under stress is achieved by decoupling the expression of different target proteins in the same cell, rather than by an increase in the total noise of each. Extrinsic noise provides the dominant contribution to the total protein noise over the total range of RyhB production rates. Stochastic simulations reproduce qualitatively key features of our observations and show that a feed-forward loop formed by transcriptional extrinsic noise, an sRNA and its target genes exhibits strong noise filtration capabilities.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Fenotipo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Regulación hacia Abajo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Hierro/metabolismo , ARN Pequeño no Traducido/metabolismo , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética , Transcripción GenéticaRESUMEN
The inherently stochastic nature of biomolecular processes is one of the main sources giving rise to cell-to-cell variations in protein and mRNA abundance, termed noise. Noise in isogenic populations can enhance survival under adverse conditions and stress, and has therefore played a fundamental role in evolution. On the other hand, noise may have detrimental effects and therefore cells must also display robustness to fluctuations and possess mechanisms of control in order to function properly. Noise can be introduced at every step in the cascade of intermediate events resulting in the production of functional proteins. While initial studies of noise focused on stochasticity introduced at the transcriptional level, recent years have witnessed a gradual shift of emphasis into the effects that post-transcriptional processes have on phenotypic noise. Here, we survey the insights that have been gained on the effects of processes that modify RNA transcript populations on phenotypic noise, including regulation by noncoding RNAs in prokaryotes and eukaryotes, alternative splicing and transcriptional interference.
Asunto(s)
Interferencia de ARN/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Animales , Humanos , ARN Mensajero/genética , ARN no Traducido/genéticaRESUMEN
The tumor suppressor BRCA2 protein plays a major role in the regulation of Rad51-catalyzed homologous recombination. BRCA2 interacts with monomeric Rad51 primarily via conserved BRC domains and coordinates the formation of Rad51 filaments at double-stranded DNA (dsDNA) breaks. A number of cancer-associated mutations in BRC4 and BRC2 domains have been reported. To elucidate their effects on homologous recombination, we studied Rad51 filament formation on single-stranded DNA and dsDNA substrates and Rad51-catalyzed strand exchange, in the presence of wild-type and mutated peptides of either BRC4 or BRC2. While the wild-type BRC2 and BRC4 peptides inhibited filament formation and, thus, strand exchange, the mutated forms decreased significantly these inhibitory effects. These results are consistent with a three-dimensional model for the interface between individual BRC repeats and Rad51. We suggest that mutations at sites crucial for the association between Rad51 and BRC domains impair the ability of BRCA2 to recruit Rad51 to dsDNA breaks, hampering recombinational repair.