SuhB is an integral part of the ribosomal antitermination complex and interacts with NusA

Dudenhoeffer, Benjamin R; Schneider, Hans; Schweimer, Kristian; Knauer, Stefan H.

doi:10.1093/nar/gkz442

Cited by 20 publications

(30 citation statements)

References 74 publications

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“…NusA and NusG are essential in E. coli (69), so we next tested NusB for its role in RNAP clustering. NusB colocalizes with RNAP clusters (70) and, together with NusA, NusG, NusE (the 30S ribosomal protein S10) and SuhB, forms a complex that prevents premature Rhodependent termination of rrn genes (71,72). When we visualized RpoC-mCherry in the ΔnusB strain, we observed a more dispersed localization pattern ( Fig.…”

Section: Rela Spotmentioning

confidence: 84%

“…3C). The central S1 motif and two K homology (KH) domains bind RNA (73,74), while the Nterminal domain (NTD) and two C-terminal acidic repeat (AR) domains interact with a variety of proteins, including multiple subunits of RNAP (75-77), NusG (78), SuhB (71,72) and the bacteriophage lambda protein N (79,80). AR2 can also interact with KH1 to block RNA binding (76,81).…”

Section: Nusa Undergoes Llps In Vitromentioning

confidence: 99%

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

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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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 nutrientrich 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. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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Section: Rela Spotmentioning

confidence: 84%

Section: Nusa Undergoes Llps In Vitromentioning

confidence: 99%

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

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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“…AT does not only play a role in the life cycle of bacteriophages but is necessary for the expression of certain bacterial genes 15,16 . In ribosomal AT, for example, RNAP pauses at an AT signal and a TAC is formed that contains Nus factors A, B, E, and G as well as further components such as ribosomal protein S4 and inositol monophosphatase SuhB [17][18][19][20] . If this TAC is only responsible for AT or also involved in posttranscriptional activities, e.g.…”

mentioning

confidence: 99%

“…NusG-CTD can interact with NusE and thus anchors the NusE:NusB:boxA complex to the RNAP 6,7,24 . NusG-mediated tethering of NusE:NusB:boxA to the RNAP might also be important for ribosomal AT 17,18 . The multidomain protein NusA ( Supplementary Fig.…”

mentioning

confidence: 99%

“…In E. coli and other γ-proteobacteria NusA has two acidic repeat (AR) domains, AR1 and AR2, at its C-terminus 35 . NusA-AR1 interacts with λN [36][37][38] , whereas NusA-AR2 serves as recruitment platform for various transcription factors such as the αCTD 39 , NusG-NTD 40 , and SuhB 17,18 . NusA is regulated via autoinhibition as NusA-AR2 binds to the KH1 domain, preventing RNA binding by NusA-SKK 39,41,42 .…”

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confidence: 99%

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NusA directly interacts with antitermination factor Q from phage λ

Dudenhoeffer

Borggraefe

Schweimer

et al. 2020

Sci Rep

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Antitermination (At) is a ubiquitous principle in the regulation of bacterial transcription to suppress termination signals. in phage λ antiterminator protein Q controls the expression of the phage's late genes with loading of λQ onto the transcription elongation complex halted at a σ-dependent pause requiring a specific DNA element. The molecular basis of λQ-dependent At and its dependence on n-utilization substance (nus) A is so far only poorly understood. Here we used solution-state nuclear magnetic resonance spectroscopy to show that the solution structure of λQ is in agreement with the crystal structure of an N-terminally truncated variant and that the 60 residues at the N-terminus are unstructured. We also provide evidence that multidomain protein nusA interacts directly with λQ via its N-terminal domain (NTD) and the acidic repeat (AR) 2 domain, with the λQ:NusA-AR2 interaction being able to release NusA autoinhibition. The binding sites for NusA-NTD and NusA-AR2 on λQ overlap and the interactions are mutually exclusive with similar affinities, suggesting distinct roles during λQ-dependent At, e.g. the λQ:nusA-ntD interaction might position nusA-ntD in a way to suppress termination, making nusA-ntD repositioning a general scheme in At mechanisms. Transcription of all cellular genomes is mediated by evolutionary related multisubunit RNA polymerases (RNAPs) 1. RNA synthesis is a discontinuous process that underlies tight regulation by various transcription factors that bind to RNAP, affecting its processivity. In Gram-negative bacteria the core RNAP consists of five subunits (2 x α, β, β' , ω). The flap region of the β subunit forms the outer wall of the RNA exit channel with the β flap tip helix (βFTH) at the top of this region being able to regulate the width of the channel, making it a key regulatory element 2-8. Antitermination (AT) is a ubiquitous mechanism to suppress termination signals and is widely used in bacteria. AT has first been described for bacteriophage λ where it controls the expression of early and late genes, being thus essential for the life cycle of the phage 9. Phage λ uses two AT mechanisms which involve either antiterminator protein N or antiterminator protein Q. In λN-dependent AT the intrinsically disordered protein N is recruited to elongating RNAP by an AT signal in the nascent RNA and forms a complex with RNAP and the Escherichia coli (E. coli) host factors N-utilization substances (Nus) A, B, E, and G 6,7. In this transcription AT complex (TAC) λN repositions NusA and remodels the βFTH, enabling the TAC to read through termination signals by preventing the formation of pause/terminator hairpins 6,7. In λQ-dependent AT protein λQ requires a λQ binding element (QBE) on the DNA for recruitment and is loaded onto RNAP halted at an adjacent sigma-dependent promoter-proximal pause site 10,11. Recent cryo electron microscopy (EM) studies of the AT mechanism of protein Q from phage 21, Q21, revealed that two Q21 proteins engage with RNAP in a Q21-TAC, one of which forms a torus at the ...

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Bacterial RNase III: Targets and physiology

Lejars

Hajnsdorf

2024

Biochimie

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SuhB is an integral part of the ribosomal antitermination complex and interacts with NusA

Cited by 20 publications

References 74 publications

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

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

NusA directly interacts with antitermination factor Q from phage λ

Bacterial RNase III: Targets and physiology

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