scholarly journals Structure of the Bacterial Ribosome at 2 Å Resolution

2020 ◽  
Author(s):  
Zoe L. Watson ◽  
Fred R. Ward ◽  
Raphaël Méheust ◽  
Omer Ad ◽  
Alanna Schepartz ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Noncovalent Interactions ◽  
Ribosomal Subunit ◽  
E Coli ◽  
Bacterial Ribosome ◽  
Domains Of Life ◽  
Chemical Resolution ◽  
70S Ribosome ◽  
A Site ◽  
First Time

AbstractContinuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 Å, based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 Å, based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

scholarly journals Structure of the bacterial ribosome at 2 Å resolution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zoe L Watson ◽  
Fred R Ward ◽  
Raphaël Méheust ◽  
Omer Ad ◽  
Alanna Schepartz ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Structure Modeling ◽  
Cryo Electron Microscopy ◽  
Bacterial Ribosome ◽  
Domains Of Life ◽  
70S Ribosome ◽  
A Site ◽  
Detailed Chemical

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

scholarly journals Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome

2020 ◽  
Vol 117 (34) ◽  
pp. 20530-20537
Author(s):  
Zahra Batool ◽  
Ivan B. Lomakin ◽  
Yury S. Polikanov ◽  
Christopher G. Bunick
Keyword(s):  
Messenger Rna ◽  
Acne Vulgaris ◽  
Thermus Thermophilus ◽  
Ribosomal Subunit ◽  
Direct Interaction ◽  
Bacterial Ribosome ◽  
70S Ribosome ◽  
A Site ◽  
Aminoacyl Transfer

Sarecycline is a new narrow-spectrum tetracycline-class antibiotic approved for the treatment of acne vulgaris. Tetracyclines share a common four-ring naphthacene core and inhibit protein synthesis by interacting with the 70S bacterial ribosome. Sarecycline is distinguished chemically from other tetracyclines because it has a 7-[[methoxy(methyl)amino]methyl] group attached at the C7 position of ring D. To investigate the functional role of this C7 moiety, we determined the X-ray crystal structure of sarecycline bound to theThermus thermophilus70S ribosome. Our 2.8-Å resolution structure revealed that sarecycline binds at the canonical tetracycline binding site located in the decoding center of the small ribosomal subunit. Importantly, unlike other tetracyclines, the unique C7 extension of sarecycline extends into the messenger RNA (mRNA) channel to form a direct interaction with the A-site codon to possibly interfere with mRNA movement through the channel and/or disrupt A-site codon–anticodon interaction. Based on our biochemical studies, sarecycline appears to be a more potent initiation inhibitor compared to other tetracyclines, possibly due to drug interactions with the mRNA, thereby blocking accommodation of the first aminoacyl transfer RNA (tRNA) into the A site. Overall, our structural and biochemical findings rationalize the role of the unique C7 moiety of sarecycline in antibiotic action.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid

1972 ◽  
Vol 130 (1) ◽  
pp. 103-110 ◽  
Author(s):  
L. P. Visentin ◽  
C. Chow ◽  
A. T. Matheson ◽  
M. Yaguchi ◽  
F. Rollin
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Soluble Fraction ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Fractionation Procedure ◽  
Additional Protein ◽  
70S Ribosome ◽  
Low Ionic Strength

1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K+ and 0.1m-Mg2+, were extracted with low-ionic-strength buffer 75–80% of the 30S proteins and 60–65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li+–EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

2022 ◽  
Vol 0 (0) ◽  
Author(s):  
V. Janett Olzog ◽  
Lena I. Freist ◽  
Robin Goldmann ◽  
Jörg Fallmann ◽  
Christina E. Weinberg
Keyword(s):  
Rna Seq ◽  
Site Specific ◽  
Pyrococcus Horikoshii ◽  
Total Rna ◽  
E Coli ◽  
Catalytic Rnas ◽  
Adapter Ligation ◽  
Domains Of Life ◽  
Cyclic Phosphate ◽  
A Site

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

scholarly journals A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1973 ◽  
Vol 133 (4) ◽  
pp. 739-747 ◽  
Author(s):  
A. Robinson ◽  
J. Sykes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Protein Loss ◽  
E Coli ◽  
Rrna Species ◽  
Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

scholarly journals Regulation of Ribosomal Protein OperonsrplM-rpsI,rpmB-rpmG, andrplU-rpmAat the Transcriptional and Translational Levels

2016 ◽  
Vol 198 (18) ◽  
pp. 2494-2502 ◽  
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Irina V. Boni
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
General Rule ◽  
Single Copy ◽  
Translation Initiation Region ◽  
Content Type ◽  
Regulatory Pathways ◽  
E Coli

ABSTRACTIt is widely assumed that in the best-characterized model bacteriumEscherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for severalE. colir-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time thein vivoregulation of three r-protein operons,rplM-rpsI,rpmB-rpmG, andrplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomallacZgene, we show here that at the translation level only one of these operons,rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, whilerpmB-rpmGandrplU-rpmAare not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression ofE. colir-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor ofrplM-rpsIexpressionin vivo, acting at a target within therplMtranslation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.

scholarly journals Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

2015 ◽  
Vol 59 (5) ◽  
pp. 2849-2854 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Borja Ochoa-Lizarralde ◽  
Neha Dhimole ◽  
Shu Zhou ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Resistance Mechanisms ◽  
Side Chain ◽  
Alternative Mode ◽  
X Ray Crystallography ◽  
Bacterial Ribosome ◽  
A Site

ABSTRACTAlthough both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in bothin vitroandin vivoactivity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to thetert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.

scholarly journals Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Norbert Izghirean ◽  
Claudia Waidacher ◽  
Clemens Kittinger ◽  
Miriam Chyba ◽  
Günther Koraimann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Parent Strain ◽  
Ribosomal Subunit ◽  
Tetracycline Resistance ◽  
E Coli ◽  
Growth Defects ◽  
Flexible Loop ◽  
Cold Sensitive ◽  
High Level

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

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