In-cell architecture of an actively transcribing-translating expressome

O’Reilly, Francis J.; Xue, Liang; Graziadei, Andrea; Sinn, Ludwig; Lenz, Swantje; Tegunov, Dimitry; Blötz, Cedric; Hagen, Wim J. H.; Cramer, Patrick; Stülke, Jörg; Mahamid, Julia; Rappsilber, Juri

doi:10.1101/2020.02.28.970111

Cited by 58 publications

(96 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our data show also that physical forces and translation is crucial for bringing about this scenario, which disappeared when in-house transcription was halted to reduce cellular crowding and ribosome engagement. This picture departs from the standard view of transcription-translation coupling, where RNAP and ribosomes get together in the so-called expressome complex (45,46) which, obviously, must operate in close proximity to the nucleoid's DNA encoding the gene(s) at stake.…”

Section: Disruption Of Intracellular Crowding Enables Xyl Transcriptsmentioning

confidence: 81%

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Kim

Goñi-Moreno

2020

Preprint

View full text Add to dashboard Cite

Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.IMPORTANCEThe transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that—similarly to computers also—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.

show abstract

Section: Disruption Of Intracellular Crowding Enables Xyl Transcriptsmentioning

confidence: 81%

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Kim

Goñi-Moreno

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…6f), marking the fi rst direct visualization of a drug bound to its target inside a cell. Th e density was absent in a 5.6 Å 70S reconstruction from untreated M. pneumoniae cells produced from processing with the older 1.0.6 Warp/M versions 36 (Fig. 6f).…”

Section: Enables the Visualization Of An Antibiotic Bound To 70s Rimentioning

confidence: 98%

“…To assess M's performance on in situ data in the strictest sense, i.e. in tilt series obtained from intact cells, we used a data set of chloramphenicol-treated Mycomplasma pneumoniae 36 . M was able to resolve the 70S ribosome at 3.7 Å (Fig.…”

Section: Enables the Visualization Of An Antibiotic Bound To 70s Rimentioning

confidence: 99%

“…Data previously used in another study 36 were re-analyzed with the release version of M. As described there, Mycoplasma pneumoniae strain M129(ATCC 29342)cells were grown on 200 mesh gold grids coated with a holey carbon support (R 2/1, Quantifoil). Cells were cultivated at 37 °C in modified Hayflick medium: 14.7 g/L Difco PPLO (Becton Dickinson, USA), 20% (v/v) Gibco horse serum (New Zealand origin, Life Technologies, USA), 100 mM HEPES-Na (pH 7.4), 1% (w/w) glucose, 0.002% (w/w) phenol red and 1,000 U/mL freshly dissolved penicillin G. Chloramphenicol (Cm; Sigma-Aldrich, USA) was added 15 minutes prior to vitrification, at a final concentration of 0.5 mg/ ml.…”

Section: Acquisition and Refinement Of M Pneumoniae In Situ Tilt Sermentioning

confidence: 99%

“…M. pneumoniae 70S ribosome-antibiotic map at 3.7 Å refi ned from in situ data with the new Warp-RELION-M pipeline We applied the Warp-RELION-M pipeline to an in situ tilt series data set36 . Th e achieved resolution reveals residue-level detail and a bound molecule of the antibiotic chloramphenicole (Cm).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Tegunov

Xue²,

Dienemann

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.Correspondence should be addressed to D.T. (dteguno@mpibpc.mpg.de), P.C. (patrick.cramer@mpibpc.mpg.de), and J.M. (julia.mahamid@embl.de). Figure 1| Th e Warp-RELION-M pipeline for frame and tilt series cryo-EM data refi nement Electron microscopy data are pre-processed on-the-fl y in Warp, which then exports particles as images or sub-tomograms. Particles are imported in RELION, where they can be subjected to a multitude of processing strategies, resulting in 3D reference maps, global particle pose alignments, and class assignments. Th e particle population encompassing all classes is then imported in M, where reference-based frame or tilt image alignments are performed simultaneously with further refi nement of particle poses and CTF parameters to improve resolution. Finally, M produces high-resolution reconstructions that can be used for model building. Alternatively, the improved alignments can be used in Warp to re-export particles for further, more accurate classifi cation in RELION.

show abstract

The promise and the challenges of cryo‐electron tomography

2020

View full text Add to dashboard Cite

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

show abstract

In-cell architecture of an actively transcribing-translating expressome

Cited by 58 publications

References 29 publications

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

The promise and the challenges of cryo‐electron tomography

Contact Info

Product

Resources

About