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1.
Mol Cell ; 78(2): 250-260.e5, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32097603

RESUMO

Structural maintenance of chromosomes (SMC) complexes organize chromosomes ubiquitously, thereby contributing to their faithful segregation. We demonstrate that under conditions of increased chromosome occupancy of the Escherichia coli SMC complex, MukBEF, the chromosome is organized as a series of loops around a thin (<130 nm) MukBEF axial core, whose length is ∼1,100 times shorter than the chromosomal DNA. The linear order of chromosomal loci is maintained in the axial cores, whose formation requires MukBEF ATP hydrolysis. Axial core structure in non-replicating chromosomes is predominantly linear (1 µm) but becomes circular (1.5 µm) in the absence of MatP because of its failure to displace MukBEF from the 800 kbp replication termination region (ter). Displacement of MukBEF from ter by MatP in wild-type cells directs MukBEF colocalization with the replication origin. We conclude that MukBEF individualizes and compacts the chromosome lengthwise, demonstrating a chromosome organization mechanism similar to condensin in mitotic chromosome formation.


Assuntos
Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Segregação de Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Mitose/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Origem de Replicação/genética , Proteínas Repressoras/ultraestrutura
2.
Proc Natl Acad Sci U S A ; 118(33)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385314

RESUMO

Structural maintenance of chromosomes (SMC) complexes contribute to chromosome organization in all domains of life. In Escherichia coli, MukBEF, the functional SMC homolog, promotes spatiotemporal chromosome organization and faithful chromosome segregation. Here, we address the relative contributions of MukBEF and the replication terminus (ter) binding protein, MatP, to chromosome organization-segregation. We show that MukBEF, but not MatP, is required for the normal localization of the origin of replication to midcell and for the establishment of translational symmetry between newly replicated sister chromosomes. Overall, chromosome orientation is normally maintained through division from one generation to the next. Analysis of loci flanking the replication termination region (ter), which demark the ends of the linearly organized portion of the nucleoid, demonstrates that MatP is required for maintenance of chromosome orientation. We show that DNA-bound ß2-processivity clamps, which mark the lagging strands at DNA replication forks, localize to the cell center, independent of replisome location but dependent on MukBEF action, and consistent with translational symmetry of sister chromosomes. Finally, we directly show that the older ("immortal") template DNA strand, propagated from previous generations, is preferentially inherited by the cell forming at the old pole, dependent on MukBEF and MatP. The work further implicates MukBEF and MatP as central players in chromosome organization, segregation, and nonrandom inheritance of genetic material and suggests a general framework for understanding how chromosome conformation and dynamics shape subcellular organization.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia
3.
Nucleic Acids Res ; 47(18): 9696-9707, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400115

RESUMO

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/química , Cromossomos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Repressoras/química
4.
Curr Genet ; 66(5): 895-899, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32300862

RESUMO

Structural maintenance of chromosomes (SMC) complexes are ancient and conserved molecular machines that organize chromosomes in all domains of life. We propose that the principles of chromosome folding needed to accommodate DNA inside a cell in an accessible form will follow similar principles in prokaryotes and eukaryotes. However, the exact contributions of SMC complexes to bacterial chromosome organization have been elusive. Recently, it was shown that the SMC homolog, MukBEF, organizes and individualizes the Escherichia coli chromosome by forming a filamentous axial core from which DNA loops emanate, similar to the action of condensin in mitotic chromosome formation. MukBEF action, along with its interaction with the partner protein, MatP, also facilitates chromosome individualization by directing opposite chromosome arms (replichores) to different cell halves. This contrasts with the situation in many other bacteria, where SMC complexes organise chromosomes in a way that the opposite replichores are aligned along the long axis of the cell. We highlight the similarities and differences of SMC complex contributions to chromosome organization in bacteria and eukaryotes, and summarize the current mechanistic understanding of the processes.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/metabolismo , Complexos Multiproteicos/metabolismo , Bactérias/genética , Bactérias/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo
5.
J Phys D Appl Phys ; 52(6): 064002, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30799881

RESUMO

Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins (PA-FPs). An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

6.
Mol Microbiol ; 99(4): 686-99, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26507787

RESUMO

In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub-optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. coli's internal organisation and functioning, and its fragility to stressful conditions.


Assuntos
Citoplasma/química , Citoplasma/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Divisão Celular , Organelas/metabolismo , Agregados Proteicos , Estresse Fisiológico , Temperatura , Viscosidade
7.
Nucleic Acids Res ; 41(13): 6544-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23644285

RESUMO

Using a single-RNA detection technique in live Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG. Importantly, the distribution of waiting times of Plac/ara-1 is indistinguishable from that of PBAD, if and only if induced by arabinose alone. Finally, RNA production under the control of PBAD is found to be a sub-Poissonian process. We conclude that inducer-dependent waiting times affect mean and cell-to-cell diversity in RNA numbers long after induction, suggesting that intake mechanisms have non-negligible effects on the phenotypic diversity of cell populations in natural, fluctuating environments.


Assuntos
Arabinose/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/biossíntese , Ativação Transcricional , Escherichia coli/metabolismo , Cinética , Iniciação da Transcrição Genética
8.
Bioinformatics ; 28(7): 1050-1, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22321700

RESUMO

SUMMARY: We present Mytoe, a tool for analyzing mitochondrial morphology and dynamics from fluorescence microscope images. The tool provides automated quantitative analysis of mitochondrial motion by optical flow estimation and of morphology by segmentation of individual branches of the network-like structure of the organelles. Mytoe quantifies several features of individual branches, such as length, tortuosity and speed, and of the macroscopic structure, such as mitochondrial area and degree of clustering. We validate the methods and apply them to the analysis of sequences of images of U2OS human cells with fluorescently labeled mitochondria. AVAILABILITY: Source code, Windows software and Manual available at http://www.cs.tut.fi/%7Esanchesr/mito SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: eero.lihavainen@tut.fi; andre.ribeiro@tut.fi.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/fisiologia , Software , Linhagem Celular Tumoral , Análise por Conglomerados , Biologia Computacional/métodos , Gráficos por Computador , Humanos , Microscopia de Fluorescência/métodos , Interface Usuário-Computador
9.
Bioinformatics ; 27(19): 2714-20, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21835771

RESUMO

MOTIVATION: Production and degradation of RNA and proteins are stochastic processes, difficulting the distinction between spurious fluctuations in their numbers and changes in the dynamics of a genetic circuit. An accurate method of change detection is key to analyze plasticity and robustness of stochastic genetic circuits. RESULTS: We use automatic change point detection methods to detect non-spurious changes in the dynamics of delayed stochastic models of gene networks at run time. We test the methods in detecting changes in mean and noise of protein numbers, and in the switching frequency of a genetic switch. We also detect changes, following genes' silencing, in the dynamics of a model of the core gene regulatory network of Saccharomyces cerevisiae with 328 genes. Finally, from images, we determine when RNA molecules tagged with fluorescent proteins are first produced in Escherichia coli. Provided prior knowledge on the time scale of the changes, the methods detect them accurately and are robust to fluctuations in protein and RNA levels. AVAILABILITY: Simulator: www.cs.tut.fi/~sanchesr/SGN/SGNSim.html CONTACT: andre.ribeiro@tut.fi SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Escherichia coli/genética , Redes Reguladoras de Genes/genética , Proteínas/genética , RNA/biossíntese , Saccharomyces cerevisiae/genética , Algoritmos , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Modelos Genéticos , RNA/genética , Processos Estocásticos
10.
J Theor Biol ; 315: 17-25, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22960571

RESUMO

In prokaryotes, the rate at which codons are translated varies from one codon to the next. Using a stochastic model of transcription and translation at the nucleotide and codon levels, we investigate the effects of the codon sequence on the dynamics of protein numbers. For sequences generated according to the codon frequencies in Escherichia coli, we find that mean protein numbers at near equilibrium differ with the codon sequence, due to the mean codon translation efficiencies, in particular of the codons at the ribosome binding site region. We find close agreement between these predictions and measurements of protein expression levels as a function of the codon sequence. Next, we investigate the effects of short codon sequences at the start/end of the RNA sequence with linearly increasing/decreasing translation efficiencies, known as slow ramps. The ramps affect the mean, but not the fluctuations, in proteins numbers by affecting the rate of translation initiation. Finally, we show that slow ramps affect the dynamics of small genetic circuits, namely, switches and clocks. In switches, ramps affect the frequency of switching and bias the robustness of the noisy attractors. In repressilators, ramps alter the robustness of periodicity. We conclude that codon sequences affect the dynamics of gene expression and genetic circuits and, thus, are likely to be under selection regarding both mean codon frequency as well as spatial arrangement along the sequence.


Assuntos
Códon/genética , Escherichia coli/genética , Redes Reguladoras de Genes/genética , Sequência de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Análise de Fourier , Regulação Bacteriana da Expressão Gênica , Cinética , Modelos Genéticos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica
11.
J Theor Biol ; 301: 83-94, 2012 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-22370562

RESUMO

Many pairs of genes in Escherichia coli are driven by closely spaced promoters. We study the dynamics of expression of such pairs of genes driven by a model at the molecule and nucleotide level with delayed stochastic dynamics as a function of the binding affinity of the RNA polymerase to the promoter region, of the geometry of the promoter, of the distance between transcription start sites (TSSs) and of the repression mechanism. We find that the rate limiting steps of transcription at the TSS, the closed and open complex formations, strongly affect the kinetics of RNA production for all promoter configurations. Beyond a certain rate of transcription initiation events, we find that the interference between polymerases correlates the dynamics of production of the two RNA molecules from the two TSS and affects the distribution of intervals between consecutive productions of RNA molecules. The degree of correlation depends on the geometry, the distance between TSSs and repressors. Small changes in the distance between TSSs can cause abrupt changes in behavior patterns, suggesting that the sequence between adjacent promoters may be subject to strong selective pressure. The results provide better understanding on the sequence level mechanisms of transcription regulation in bacteria and may aid in the genetic engineering of artificial circuits based on closely spaced promoters.


Assuntos
Escherichia coli/genética , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , RNA Bacteriano/biossíntese , Processos Estocásticos , Sítio de Iniciação de Transcrição
12.
BMC Bioinformatics ; 12: 121, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21521517

RESUMO

BACKGROUND: In prokaryotes, transcription and translation are dynamically coupled, as the latter starts before the former is complete. Also, from one transcript, several translation events occur in parallel. To study how events in transcription elongation affect translation elongation and fluctuations in protein levels, we propose a delayed stochastic model of prokaryotic transcription and translation at the nucleotide and codon level that includes the promoter open complex formation and alternative pathways to elongation, namely pausing, arrests, editing, pyrophosphorolysis, RNA polymerase traffic, and premature termination. Stepwise translation can start after the ribosome binding site is formed and accounts for variable codon translation rates, ribosome traffic, back-translocation, drop-off, and trans-translation. RESULTS: First, we show that the model accurately matches measurements of sequence-dependent translation elongation dynamics. Next, we characterize the degree of coupling between fluctuations in RNA and protein levels, and its dependence on the rates of transcription and translation initiation. Finally, modeling sequence-specific transcriptional pauses, we find that these affect protein noise levels. CONCLUSIONS: For parameter values within realistic intervals, transcription and translation are found to be tightly coupled in Escherichia coli, as the noise in protein levels is mostly determined by the underlying noise in RNA levels. Sequence-dependent events in transcription elongation, e.g. pauses, are found to cause tangible effects in the degree of fluctuations in protein levels.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Biossíntese de Proteínas , Transcrição Gênica , Códon , RNA Polimerases Dirigidas por DNA/metabolismo , Sequências Reguladoras de Ácido Nucleico , Ribossomos/metabolismo , Processos Estocásticos
14.
Nat Commun ; 12(1): 6721, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795302

RESUMO

Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Proteína de Transporte de Acila/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Ligação Proteica , Proteínas Repressoras/genética
15.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34585666

RESUMO

Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualization and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. These data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time.


Assuntos
Ligação Competitiva , Proteínas Cromossômicas não Histona/química , DNA Topoisomerase IV/química , Proteínas de Escherichia coli/química , Escherichia coli/química
16.
Elife ; 82019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397672

RESUMO

The chromosomal replication origin region (ori) of characterised bacteria is dynamically positioned throughout the cell cycle. In slowly growing Escherichia coli, ori is maintained at mid-cell from birth until its replication, after which newly replicated sister oris move to opposite quarter positions. Here, we provide an explanation for ori positioning based on the self-organisation of the Structural Maintenance of Chromosomes complex, MukBEF, which forms dynamically positioned clusters on the chromosome. We propose that a non-trivial feedback between the self-organising gradient of MukBEF complexes and the oris leads to accurate ori positioning. We find excellent agreement with quantitative experimental measurements and confirm key predictions. Specifically, we show that oris exhibit biased motion towards MukBEF clusters, rather than mid-cell. Our findings suggest that MukBEF and oris act together as a self-organising system in chromosome organisation-segregation and introduces protein self-organisation as an important consideration for future studies of chromosome dynamics.


Assuntos
Segregação de Cromossomos , Escherichia coli/genética , Movimento (Física) , Origem de Replicação , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Análise Espacial
17.
Sci Rep ; 7(1): 10588, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28878283

RESUMO

Cell-to-cell variability in cellular components generates cell-to-cell diversity in RNA and protein production dynamics. As these components are inherited, this should also cause lineage-to-lineage variability in these dynamics. We conjectured that these effects on transcription are promoter initiation kinetics dependent. To test this, first we used stochastic models to predict that variability in the numbers of molecules involved in upstream processes, such as the intake of inducers from the environment, acts only as a transient source of variability in RNA production numbers, while variability in the numbers of a molecular species controlling transcription of an active promoter acts as a constant source. Next, from single-cell, single-RNA level time-lapse microscopy of independent lineages of Escherichia coli cells, we demonstrate the existence of lineage-to-lineage variability in gene activation times and mean RNA production rates, and that these variabilities differ between promoters and inducers used. Finally, we provide evidence that this can be explained by differences in the kinetics of the rate-limiting steps in transcription between promoters and induction schemes. We conclude that cell-to-cell and consequent lineage-to-lineage variability in RNA and protein numbers are both promoter sequence-dependent and subject to regulation.


Assuntos
Regulação da Expressão Gênica , Transcrição Gênica , Algoritmos , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , RNA Bacteriano/genética , Ativação Transcricional
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