Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of ...conventional light microscopy (200–300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia coli with a spatial resolution of ∼40 nm. In cells growing rapidly in nutrient-rich media, we find that RNAP is organized in 2–8 bands. The band number scaled directly with cell size (and so with the chromosome number), and bands often contained clusters of >70 tightly packed RNAPs (possibly engaged on one long ribosomal RNA operon of 6000 bp) and clusters of such clusters (perhaps reflecting a structure like the eukaryotic nucleolus where many different ribosomal RNA operons are transcribed). In nutrient-poor media, RNAPs were located in only 1–2 bands; within these bands, a disproportionate number of RNAPs were found in clusters containing ∼20–50 RNAPs. Apart from their importance for bacterial transcription, our studies pave the way for molecular-level analysis of several cellular processes at the nanometer scale.
Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, ...and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ70. The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription.
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Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used ...superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.
Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts ...are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm.
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Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Toward ...identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid is not a physical barrier for protein diffusion but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58%–99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome crowding likely has important implications for the function of all DNA-binding proteins.
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•Protein motion was compared between unperturbed cells and DNA-free cells•Protein mobility was dictated by DNA interactions rather than molecular weight•The nucleoid is not a physical barrier for protein diffusion•The proteins studied spend most (58%–99%) of their search time bound to DNA
To understand how DNA-binding proteins find their target sites, Stracy et al. tracked the motion of 11 diverse proteins in living Escherichia coli. By comparing protein behavior in normal cells and cells without chromosomes, they showed that the DNA-binding proteins spend most of their search time bound to DNA.
•Single-molecule FRET can be applied inside live cells.•Optimal labeling, internalization and imaging strategies are being developed.•smFRET detection has been demonstrated in both microbial and ...mammalian systems.•Biomolecular structure and dynamics can be studied.
Single-molecule Förster resonance energy transfer (smFRET) serves as a molecular ruler that is ideally posed to study static and dynamic heterogeneity in living cells. Observing smFRET in cells requires appropriately integrated labeling, internalization and imaging strategies, and significant progress has been made towards that goal. Pioneering studies have demonstrated smFRET detection in both prokaryotic and eukaryotic systems, using both wide-field and confocal microscopies, and have started to answer exciting biological questions. We anticipate that future technical developments will open the door to smFRET for the study of structure, conformational changes and kinetics of biomolecules in living cells.
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial ...transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.
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•E. coli RNA polymerase pauses during initial transcription at lac promoters•Initiation pausing lasts for ∼20 s and occurs at the transition from 6- to 7-nt RNA•Region 3.2 of σ70 is the main protein element controlling pausing•Pausing is likely to be controlled further by a complex set of determinants
Initial transcription involves synthesis of short RNAs that are either released or extended to allow promoter escape; such mechanisms are unclear due to heterogeneity. Duchi et al. used single-molecule fluorescence to visualize transcription in real time and discovered a long pause after synthesis of a 6-mer RNA.
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.
•Super-resolution imaging and tracking of RNA polymerase RNAP in living bacteria.•RNAP tracking reports on its intracellular mobility and spatial distribution.•RNAP spatial and diffusion profile is ...sensitive to antibiotics and growth media.•RNAPs on highly expressed genes are found in clusters at the nucleoid periphery.•RNAP spends most of its promoter search time bound non-specifically to DNA.
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription.
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