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.RNA polymerase | transcription | superresolution | single-molecule tracking | protein-DNA interactions
SMC (structural maintenance of chromosome) proteins act ubiquitously in chromosome processing. In Escherichia coli, the SMC complex MukBEF plays roles in chromosome segregation and organization. We used single-molecule millisecond multicolor fluorescence microscopy of live bacteria to reveal that a dimer of dimeric fluorescent MukBEF molecules acts as the minimal functional unit. On average, 8 to 10 of these complexes accumulated as "spots" in one to three discrete chromosome-associated regions of the cell, where they formed higher-order structures. Functional MukBEF within spots exchanged with freely diffusing complexes at a rate of one complex about every 50 seconds in reactions requiring adenosine triphosphate (ATP) hydrolysis. Thus, by functioning in pairs, MukBEF complexes may undergo multiple cycles of ATP hydrolysis without being released from DNA, analogous to the behavior of well-characterized molecular motors.
Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.single-molecule tracking | super-resolution microscopy | DNA damage response | protein-DNA interaction | cytosolic diffusion A ll cellular organisms rely on complex DNA repair mechanisms for faithful chromosome replication and maintenance of their genome integrity (1). The variety of DNA lesions requires modular repair pathways that carry out damage recognition, damage removal, repair synthesis, and ligation in sequential steps catalyzed by a series of enzymes. However, all repair pathway steps need to be precisely balanced to avoid accumulation of DNA intermediates that are typically more mutagenic and toxic than the original lesion (2). Rapid processing of gapped and nicked intermediates is particularly crucial (3) because they provoke lethal double-strand breaks upon encountering replication forks (4); a single such break can lead to chromosome loss and cell death.Despite extensive genetic, biochemical, and biophysical studies (1), the molecular organization of DNA repair in vivo remains unclear. Most of our mechanistic understanding relies on in vitro ensemble studies, which cannot replicate the cellular environment and stochastic nature of chemical reactions. By avoiding ensembleaveraging, single-molecule experiments have revolutionized the study of protein-DNA interactions in vitro, but extension of these powerful concepts to DNA repair measurements in living cells remains an open goal. Early in vivo work focused on the mean behavior of cell populations and could not examine functionally important heterogeneity, such as the variation in protein copy numbers between cells and over time (5, 6). Such variation can lead to different repair rates across genetically identical cells and may dera...
Cells rely on the precise action of proteins that detect and repair DNA damage. However, gene expression noise causes fluctuations in protein abundances that may compromise repair. For the Ada protein in Escherichia coli, which induces its own expression upon repairing DNA alkylation damage, we found that undamaged cells on average produce one Ada molecule per generation. Because production is stochastic, many cells have no Ada molecules and cannot induce the damage response until the first expression event occurs, sometimes delaying the response for generations. This creates a subpopulation of cells with increased mutation rates. Non-genetic variation in protein abundances thus leads to genetic heterogeneity in the population. Our results further suggest that cells balance reliable repair against toxic side-effects of abundant DNA repair proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.