Visualizing the transfer-messenger RNA as the ribosome resumes translation

Bacterial ribosomes that are stalled on mRNAs lacking a stop codon can be rescued by a process called ‘transtranslation' that involves the ribonucleoprotein complex tmRNA–SmpB. This cryo-EM study, and the copublished study by Weis et al, reveal how translation on tmRNA is resume

Dynamical features of the Plasmodium falciparum ribosome during translation

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials

Escherichia coli NusG Links the Lead Ribosome with the Transcription Elongation Complex

It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG

Escherichia coli NusG Links the Lead Ribosome with the Transcription Elongation Complex

It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG

Vers une meilleure compréhension des interactions ARN/ligands (méthodes, outils et applications)

L objectif de cette thèse est d essayer de mieux comprendre les interactions ARN/ligand, et plus particulièrement, les interactions entre le site-A de l ARN ribosomal bactérien 16S et différents antibiotiques de la famille des aminoglycosides, en utilisant les données théoriques et expérimentales des simulations de DM et de bases de données structurales. Ainsi, un ensemble d outils et de méthodes ont été développés visant, entre autres, à mettre en relation les données des simulations de DM avec les données structurales issues des expériences de diffraction de rayons X. Cette mise en relation permet de valider les données des simulations de DM ainsi que de combler certaines des carences des structures expérimentales. En effet, ce travail prend en compte un certain nombre d aspects de développement et d aspects méthodologiques comprenant, entre autres, la conception du web service SwS, l élaboration de méthodes de détection des positions des atomes d hydrogène et de techniques de cartographie des sites de solvatation basées sur les données expérimentales (structures RX) et théoriques (simulations de DM). Les outils et méthodes développés ont été mis à profit dans l étude de différents complexes site-A/aminoglycosides, révélant, entre autres, certains de leurs aspects dynamiques intéressants.The objective of this thesis is to try to better understand RNA/ligands interactions and especially the interactions between the A-site, part of the bacterial 16S ribosomal RNA, and various antibiotics of the aminoglycosides family, using theoretical MD simulations data and experimental structural databases. Thus, a set of tools and methods have been developed aiming, among other things, to link data from MD simulations with the structural data derived from X-ray diffraction experiments. This link allows validating MD simulations data and addressing some of experimental structures shortcomings. Indeed, this work involves a number of development and methodological aspects including, among other things, the programming of the SwS web service, the development of methods for detecting hydrogen atoms positions and mapping techniques of solvation sites based on experimental (structures RX) and theoretical data (MD simulations). The tools and methods developed have been used in the study of various A-site/aminoglycosides complexes revealing, among other things, some of their interesting dynamic aspects.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

SwS: a solvation web service for nucleic acids.

International audienceSUMMARY: SwS, based on a statistical analysis of crystallographic structures deposited in the NDB, is designed to provide an exhaustive overview of the solvation of nucleic acid structural elements through the generation of three-dimensional solvent density maps. A first version (v1.0) of this web service focuses on the interaction of DNA, RNA and hybrid base pairs linked by two or three hydrogen bonds with water, cations and/or anions. Data provided by SwS are updated on a weekly basis and can be used by: (i) those involved in molecular dynamics simulation studies for validation purposes; (ii) crystallographers for help in the interpretation of solvent density maps; and all those involved in (iii) drug design and, more generally, in (iv) nucleic acid structural studies. SwS provides also statistical data related to the frequency of occurrence of different types of base pairs in crystallographic structures and the conformation of the involved nucleotides. This web service has been designed to allow a maximum of flexibility in terms of queries and has also been developed with didactic considerations in mind. AVAILABILITY: http://www-ibmc.u-strasbg.fr/arn/sws.html

Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis

International audienceKinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation

Estimation of the 3D Variance-Covariance Map in Cryo-Electron Microscopy

Single-particle cryo-electron microscopy (cryo-EM) has recently become an important tool for the study of macromolecular structures at high resolution. One challenge, however, is that the data collected are intrinsically heterogeneous, which limits the resolution that can be potentially achieved. One way to address the heterogeneity problem is via computation of the covariance matrix, which captures the correlation between every pair of voxels, thereby revealing the variability and co-variability of the underlying structures, in terms of spatial location and the type of structural change. Specifically, we propose an iterative approach in the image domain to the estimation of the covariance matrix from cryo-EM single-particle images. Although this type of approach is commonly perceived as being slow, it has two important mitigating advantages: constraints on the solution can be easily imposed; and the solution domain can be tailored to have arbitrary shape and size, thereby considerably reducing the number of unknowns, which grows quadratically with the size of the volume. We obtained encouraging results on an experimental data set with 29,000 projections of a 43S ribosomal pre-initiation complex