scholarly journals Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB

2021 ◽  
Author(s):  
Yannan Tian ◽  
Fuxing Zeng ◽  
Adrika Raybarman ◽  
Amy Carruthers ◽  
Qingrong Li ◽  
...  
Keyword(s):  
Cell Survival ◽  
Ribosomal Subunit ◽  
Translation System ◽  
Rna Repair ◽  
Damage Site ◽  
Specific Recognition ◽  
Bacterial System ◽  
Peptide Release ◽  
70S Ribosome ◽  
Insight Into

AbstractIn bacteria, rescue of stalled ribosomes due to 3’-truncated mRNAs is carried out by the ubiquitous trans-translation system as well as alternative ribosome-rescue factors such as ArfA and ArfB. It is unclear, however, how the stalled ribosomes caused by ribosomal damages are rescued. Here, we report that a bacterial system composed of PrfH and RtcB not only rescues a stalled ribosome resulting from a specific damage in the decoding center but also repairs the damage afterwards. Peptide release assays reveal that PrfH is only active with the damaged ribosome, but not with the intact one. A 2.55-angstrom cryo-EM structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into specific recognition of the damage site by PrfH. RNA repair assays demonstrate that PrfH-coupled RtcB efficiently repairs the damaged 30S ribosomal subunit, but not the damaged tRNAs. Thus, our studies have uncovered a biological operation by a pair of bacterial enzymes, aiming to reverse the potentially lethal damage inflicted by an invading ribotoxin for cell survival.

scholarly journals Structure of a canonical RNase YoeB bound to the ribosome

2014 ◽  
Vol 70 (a1) ◽  
pp. C207-C207
Author(s):  
Yun Chen ◽  
Shu Feng ◽  
Katsuhiko Kamada ◽  
Han Wang ◽  
Kai Tang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Base Catalysis ◽  
Mass Spectrometry Data ◽  
Catalytic Core ◽  
General Base ◽  
Rna Hydrolysis ◽  
70S Ribosome ◽  
A Site ◽  
Insight Into

As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'–phosphate at the newly formed 3' end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

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%).

Platelets in tissue repair: control of apoptosis and interactions with regenerative cells

Blood ◽  
2013 ◽  
Vol 122 (15) ◽  
pp. 2550-2554 ◽  
Author(s):  
Meinrad Gawaz ◽  
Sebastian Vogel
Keyword(s):  
Cell Survival ◽  
Tissue Repair ◽  
Molecular Mechanisms ◽  
Healing Process ◽  
Critical Role ◽  
Cellular Migration ◽  
Tissue Repair And Regeneration ◽  
Primary Hemostasis ◽  
Regenerative Cells ◽  
Insight Into

Abstract Besides mediating primary hemostasis and thrombosis, platelets play a critical role in tissue repair and regeneration. They regulate fundamental mechanisms involved in the healing process including cellular migration, proliferation, and angiogenesis. Control of apoptosis/cell survival and interaction with progenitor cells, which are clinically relevant but poorly understood aspects of platelets in tissue repair, will be highlighted in this review. Gaining deeper insight into the less well-characterized molecular mechanisms is necessary to develop new therapeutic platelet-based options.

scholarly journals The structural basis for inhibition of ribosomal translocation by viomycin

2020 ◽  
Vol 117 (19) ◽  
pp. 10271-10277
Author(s):  
Ling Zhang ◽  
Ying-Hui Wang ◽  
Xing Zhang ◽  
Laura Lancaster ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
23S Rrna ◽  
Subunit Dissociation ◽  
Classical State ◽  
State Transfer ◽  
70S Ribosome ◽  
Intersubunit Rotation

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

Cryo-EM structure of 90S small ribosomal subunit precursors in transition states

Science ◽  
2020 ◽  
Vol 369 (6510) ◽  
pp. 1477-1481 ◽  
Author(s):  
Yifei Du ◽  
Weidong An ◽  
Xing Zhu ◽  
Qi Sun ◽  
Jia Qi ◽  
...  
Keyword(s):  
Structural Changes ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Rna Degradation ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
Nuclear Exosome ◽  
Assembly Intermediate ◽  
Insight Into

The 90S preribosome is a large, early assembly intermediate of small ribosomal subunits that undergoes structural changes to give a pre-40S ribosome. Here, we gained insight into this transition by determining cryo–electron microscopy structures of Saccharomyces cerevisiae intermediates in the path from the 90S to the pre-40S. The full transition is blocked by deletion of RNA helicase Dhr1. A series of structural snapshots revealed that the excised 5′ external transcribed spacer (5′ ETS) is degraded within 90S, driving stepwise disassembly of assembly factors and ribosome maturation. The nuclear exosome, an RNA degradation machine, docks on the 90S through helicase Mtr4 and is primed to digest the 3′ end of the 5′ ETS. The structures resolved between 3.2- and 8.6-angstrom resolution reveal key intermediates and the critical role of 5′ ETS degradation in 90S progression.

What we have learned from ribosome structures

2008 ◽  
Vol 36 (4) ◽  
pp. 567-574 ◽  
Author(s):  
V. Ramakrishnan
Keyword(s):  
High Resolution ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
30S Subunit ◽  
High Resolution Structure ◽  
70S Ribosome ◽  
Year 2000 ◽  
Resolution Structure

The determination of the high-resolution structures of ribosomal subunits in the year 2000 and of the entire ribosome a few years later are revolutionizing our understanding of the role of the ribosome in translation. In the present article, I summarize the main contributions from our laboratory to this worldwide effort. These include the determination of the structure of the 30S ribosomal subunit and its complexes with antibiotics, the role of the 30S subunit in decoding, and the high-resolution structure of the entire 70S ribosome complexed with mRNA and tRNA.

scholarly journals Mechanism of ribosome rescue by ArfA and RF2

2016 ◽  
Author(s):  
Gabriel Demo ◽  
Egor Svidritskiy ◽  
Rohini Madireddy ◽  
Ruben Diaz-Avalos ◽  
Timothy Grant ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Stop Codon ◽  
Release Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
C Terminus ◽  
Peptide Release ◽  
N Terminus ◽  
70S Ribosome ◽  
A Site

AbstractArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

scholarly journals Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

2021 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Attilio Fabbretti ◽  
Pohl Milon ◽  
Xu Han ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Resistance Mechanisms ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
A Site ◽  
Insight Into ◽  
Major Target

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

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 Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo

2020 ◽  
Author(s):  
Louis-Valentin Méteignier ◽  
Rabea Ghandour ◽  
Aude Zimmerman ◽  
Lauriane Kuhn ◽  
Jörg Meurer ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Higher Plants ◽  
Physiological Effects ◽  
Ribosomal Assembly ◽  
Transcription Termination Factor ◽  
Protein Interactome ◽  
70S Ribosome ◽  
Insight Into

ABSTRACTThe mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ~30 amino acids. They are found in metazoans and plants where they localize to mitochondria or chloroplasts. In higher plants, the mTERF family comprises ~30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

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