RESUMO
Loading of the ring-shaped Mcm2-7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Manutenção de Minicromossomo/isolamento & purificação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Núcleo Celular/metabolismo , Complexo Mediador/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , TATA Box/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica/genéticaRESUMO
Understanding the pathway and kinetic mechanisms of transcription initiation is essential for quantitative understanding of gene regulation, but initiation is a multistep process, the features of which can be obscured in bulk analysis. We used a multiwavelength single-molecule fluorescence colocalization approach, CoSMoS, to define the initiation pathway at an activator-dependent bacterial σ(54) promoter that recapitulates characteristic features of eukaryotic promoters activated by enhancer binding proteins. The experiments kinetically characterize all major steps of the initiation process, revealing heretofore unknown features, including reversible formation of two closed complexes with greatly differing stabilities, multiple attempts for each successful formation of an open complex, and efficient release of σ(54) from the polymerase core at the start of transcript synthesis. Open complexes are committed to transcription, suggesting that regulation likely targets earlier steps in the mechanism. CoSMoS is a powerful, generally applicable method to elucidate the mechanisms of transcription and other multistep biochemical processes.
Assuntos
Regiões Promotoras Genéticas , Fator sigma/metabolismo , Análise Espectral/métodos , Transcrição Gênica , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Dados de Sequência Molecular , Salmonella typhimurium/metabolismoRESUMO
Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.
Assuntos
Proteínas de Escherichia coli , RNA Bacteriano , RNA Bacteriano/metabolismo , Cinética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Transcrição Gênica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ70; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to â¼1,000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.
Assuntos
RNA Polimerases Dirigidas por DNA , DNA , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiões Promotoras Genéticas , Óperon/genética , Transcrição Gênica , Fator sigma/genética , DNA Bacteriano/metabolismoRESUMO
During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC (origin recognition complex) can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA-binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally controlled DNA sliding of helicase-loading intermediates and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA-binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely oriented Mcm2-7 helicases to ensure bidirectional DNA replication.
Assuntos
Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , DNA/genética , DNA/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismoRESUMO
Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.
Assuntos
Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Fosforilação , Origem de Replicação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Quinases Ciclina-Dependentes/metabolismoRESUMO
Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch-junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin-actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament-Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.
Assuntos
Fatores de Despolimerização de Actina , Fator de Maturação da Glia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Fator de Maturação da Glia/metabolismo , Microscopia de Fluorescência , Imagem Individual de MoléculaRESUMO
Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.
Assuntos
Chaperona BiP do Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Camundongos , Dobramento de ProteínaRESUMO
In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Cinética , Modelos Teóricos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos , Fatores de Elongação da Transcrição/genéticaRESUMO
Microtubule-based active matter provides insight into the self-organization of motile interacting constituents. We describe several formulations of microtubule-based 3D active isotropic fluids. Dynamics of these fluids is powered by three types of kinesin motors: a processive motor, a non-processive motor, and a motor which is permanently linked to a microtubule backbone. Another modification uses a specific microtubule crosslinker to induce bundle formation instead of a non-specific polymer depletant. In comparison to the already established system, each formulation exhibits distinct properties. These developments reveal the temporal stability of microtubule-based active fluids while extending their reach and the applicability.
Assuntos
Longevidade , Microtúbulos , CinesinasRESUMO
The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors' interactions with RNAP and how they function without cross-interference are unclear. In Escherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3-0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Benzenossulfonatos , Sítios de Ligação , Carbocianinas , Simulação por Computador , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes , Modelos Genéticos , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Imagem Individual de Molécula , Fatores de Tempo , Elongação da Transcrição GenéticaRESUMO
Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fator sigma/metabolismo , Transcrição Gênica , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Cinética , Lasers , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Fotodegradação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Moldes Genéticos , Fatores de Tempo , Elongação da Transcrição GenéticaRESUMO
Multi-wavelength single molecule fluorescence microscopy is a valuable tool for clarifying transcription mechanisms, which involve multiple components and intermediates. Here we describe methods for the analysis and interpretation of such single molecule data. The methods described include those for image alignment, drift correction, spot discrimination, as well as robust methods for analyzing single-molecule binding and dissociation kinetics that account for non-specific binding and photobleaching. Finally, we give an example of the use of the resulting data to extract the kinetic mechanism of promoter binding by a bacterial RNA polymerase holoenzyme.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Nanotecnologia/métodos , Regiões Promotoras Genéticas , Transcrição Gênica , DNA/química , RNA Polimerases Dirigidas por DNA/química , Cinética , Microscopia de Fluorescência , Modelos Teóricos , FotodegradaçãoRESUMO
Sequence-specific DNA binding proteins must quickly bind target sequences, despite the enormously larger amount of nontarget DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by facilitated diffusion (FD). In FD, a protein first binds to nontarget DNA and then reaches the target by a 1D sliding search. We tested whether Escherichia coli σ(54)RNA polymerase reaches a promoter by FD using the colocalization single-molecule spectroscopy (CoSMoS) multiwavelength fluorescence microscopy technique. Experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and nonpromoter DNAs measured in the same sample under identical conditions. Binding to a nonpromoter DNA was much slower than binding to a promoter-containing DNA of the same length, indicating that the detected nonspecific binding events are not on the pathway to promoter binding. Truncating one of the DNA segments flanking the promoter to a length as short as 7 bp or lengthening it to ~3,000 bp did not alter the promoter-specific binding rate. These results exclude FD over distances corresponding to the length of the promoter or longer from playing any significant role in accelerating promoter search. Instead, the data support a direct binding mechanism, in which σ(54)RNA polymerase reaches the local vicinity of promoters by 3D diffusion through solution, and suggest that binding may be accelerated by atypical structural or dynamic features of promoter DNA. Direct binding explains how polymerase can quickly reach a promoter, despite occupancy of promoter-flanking DNA by bound proteins that would impede FD.
Assuntos
DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Difusão , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cinética , Microscopia de Fluorescência/métodos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Ligação Proteica , RNA Polimerase Sigma 54/genéticaRESUMO
Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/química , Actinas/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Cinética , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/ultraestruturaRESUMO
Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.
Assuntos
Conformação de Ácido Nucleico , Sítios de Splice de RNA/genética , Splicing de RNA/fisiologia , Spliceossomos/fisiologia , Sequência de Bases , Primers do DNA/genética , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Dados de Sequência Molecular , Oligonucleotídeos/genética , Saccharomyces cerevisiae , Análise EspectralRESUMO
The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Óperon Lac , Repressores Lac/genética , Lactose/metabolismo , Microscopia de Fluorescência/métodos , Modelos Genéticos , TermodinâmicaRESUMO
Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Trimetoprima/química , Carbocianinas/química , Corantes Fluorescentes/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Fótons , Trimetoprima/síntese químicaRESUMO
As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."