RESUMEN
Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.
Asunto(s)
Escherichia coli/fisiología , Flagelos/metabolismo , Locomoción/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas Bacterianas , Proteínas de Escherichia coli , Unión Proteica/fisiologíaRESUMEN
Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.
Asunto(s)
Gránulos Citoplasmáticos/química , ARN Helicasas DEAD-box/química , Orgánulos/química , Transición de Fase , Secuencia de Aminoácidos , Núcleo Celular/química , Núcleo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN/química , ADN/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Metilación , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Orgánulos/metabolismo , Concentración Osmolar , Homología de Secuencia de Aminoácido , Electricidad Estática , Imagen de Lapso de Tiempo , Temperatura de TransiciónRESUMEN
DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (â¼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Secuencia de Bases , Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Desnaturalización de Ácido Nucleico , Especificidad por SustratoRESUMEN
Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new technique, solution wide-field imaging (SWiFi) of diffusing molecules, as an alternative to the existing methods. SWiFi is a simple extension to existing objective-type TIRF microscopes that allows wide-field observations of fast-diffusing molecules down to single fluorophores without the need of tethering the molecules to the surface. We demonstrate that SWiFi enables high-throughput ratiometric measurements with several thousands of individual data points per minute on double-stranded DNA standard (dsDNA) samples containing Förster resonance energy transfer pairs. We further display the capabilities of SWiFi by reporting on mobility and ratiometric characterization of fluorescent nanodiamonds, DNA Holliday junctions, and protein-DNA interactions. The ability of SWiFi for high-throughput, ratiometric measurements of fast-diffusing species renders it a valuable tool for the single-molecule research community by bridging between confocal and TIRF detection geometries in a simple and efficient way.
Asunto(s)
Colorantes Fluorescentes/química , Imagen Individual de Molécula/métodos , ADN Cruciforme/química , ADN Cruciforme/genética , ARN Polimerasas Dirigidas por ADN/genética , Difusión , Transferencia Resonante de Energía de Fluorescencia , Regiones Promotoras Genéticas/genéticaRESUMEN
Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of â¼8 µm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.
Asunto(s)
Bacterias/genética , ARN de Transferencia/metabolismo , ARN/metabolismo , Bacterias/metabolismo , Difusión , Escherichia coli/genética , Escherichia coli/metabolismo , Imagen Molecular , Biosíntesis de Proteínas , Transporte de ARN , ARN BacterianoRESUMEN
Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/análisis , ARN Polimerasas Dirigidas por ADN/metabolismo , Electroporación/métodos , Escherichia coli/metabolismo , Fluorescencia , Supervivencia Celular , ARN Polimerasas Dirigidas por ADN/química , Difusión , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , GlicerolRESUMEN
Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.
Asunto(s)
Electroporación/métodos , Colorantes Fluorescentes/metabolismo , Microscopía Fluorescente/métodos , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , ADN Bacteriano/metabolismo , ADN de Hongos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.