Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

https://www.pnas.org/content/pnas/117/31/18540.full.pdf

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation

Bacterial ribosome biogenesis has been an active area of research for more than 30 years and has served as a test-bed for the development of new biochemical, biophysical and structural techniques to understand macromolecular assembly generally. Recent work inspecting the process in vivo has advanced our understanding of the role of ribosome biogenesis factors, the co-transcriptional nature of assembly, the kinetics of the process under sub-optimal conditions, and the rRNA folding and ribosome protein binding pathways. Additionally, new structural work enabled by single-particle electron microscopy has helped to connect in vitro ribosomal protein binding maps to the underlying RNA. This review summarizes the state of these in vivo studies, provides a kinetic model for ribosome assembly under sub-optimal conditions, and describes a framework to compare newly emerging assembly intermediate structures.This article is part of the themed issue 'Perspectives on the ribosome'.

https://royalsocietypublishing.org/doi/pdf/10.1098/rstb.2016.0181

Structure and dynamics of bacterial ribosome biogenesis

Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life.

https://biorxiv.org/content/10.1101/2020.03.16.994491v1.full.pdf

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins’ folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S–RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

https://www.mdpi.com/1422-0067/24/3/2118/pdf?version=1675128137

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus

An extended catalogue of ncRNAs in Streptomyces coelicolor reporting abundant tmRNA, RNase-P RNA and RNA fragments derived from pre-ribosomal RNA leader sequences

The ribosome is a structurally and functionally conserved macromolecular machine responsible for translating messenger ribonucleic acid (mRNA) into proteins. Composed of two independently assembled subunits, the bacterial ribosome is approximately 2.5 MDa in size. The process of translation, catalysed by the ribosome, is central to gene expression in the cell. Correspondingly, the regulation of ribosome biogenesis is paramount to cell viability, growth and differentiation. Moreover, the ribosome is the target of numerous clinically relevant therapeutic agents, several of which are known to affect ribosome assembly. Although, in vitro reconstitution of bacterial ribosomal subunits can be achieved using purified ribosomal RNA (rRNA) and r-protein components, the process occurs at nonphysiological conditions. It is suggested that a specific order of processing and assembly events and regulatory factors is required to achieve fully functional particles. This article focuses on the protein factors that are involved in the process of bacterial ribosome biogenesis.



Key concepts: 



This article focuses on the protein factors that are involved in the process of bacterial ribosome biogenesis.






Keywords:

assembly;
biogenesis;
ribosome;
RNA;
translation;
bacteria

Factor-Mediated Ribosome Assembly in Bacteria

Polycystic Ovary Syndrome (PCOS) remains a major health problem in women, characterized by hyperinsulinemia, hyperandrogenism, menstrual irregularities, and long-term metabolic disturbances. Ovarian steroidogenesis is one of the key signalling pathways for follicular development. Due to the overexpression of the key enzymes (CYP 17A1, CYP 11A1, CYP 1A1, CYP 19A1, STAR and HSD17B1) present in this pathway, androgen is converted more efficiently to testosterone, causing hyperandrogenism and hyperinsulinemia. In this study, virtual screening of 203 phytochemicals collected from 44 plants were used as ligands to identify potent inhibitors for each of the promising targets of PCOS. Subsequently, a multitargeted docking operation was carried out, which rendered the 22 best docking poses. In-silico ADME and drug-likeness prediction of 9 of these 22 phytochemicals showed good pharmacokinetic properties, having high gastrointestinal absorption, blood-brain barrier permeability, and less toxic. Six phytochemicals: Chlorogenin, Hispaglabridin B, Ruscogenin, Glabridin, Degulin, and Diosgenin were evaluated as the best potential inhibitors for HSD17B1, CYP11A1, STAR, CYP1A1 CYP19A1 and CYP17A1, PCOS proteins, respectively. This study is believed to aid in the identification and future lead optimization for the natural treatment of PCOS.

Lead Identification against Polycystic Ovary Syndrome using Molecular Docking Approach

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Factor-Mediated Ribosome Assembly in Bacteria

Lead Identification against Polycystic Ovary Syndrome using Molecular Docking Approach