scholarly journals Cryo-Electron Microscopy Visualization of a Large Insertion in the 5S ribosomal RNA of the Extremely Halophilic Archaeon Halococcus morrhuae

2020 ◽  
Author(s):  
Madhan R Tirumalai ◽  
Jason T Kaelber ◽  
Donghyun R Park ◽  
Quyen Tran ◽  
George E Fox
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Rna ◽  
Large Subunit ◽  
Insertion Site ◽  
5S Rrna ◽  
Extreme Halophile ◽  
Cryo Electron Microscopy ◽  
Junction Site ◽  
Nucleotide Insertion ◽  
Extremely Halophilic Archaeon

AbstractThe extreme halophile Halococcus morrhuae (ATCC® 17082) contains a 108-nucleotide insertion in its 5S rRNA. Large rRNA expansions in Archaea are rare. This one almost doubles the length of the 5S rRNA. In order to understand how such an insertion is accommodated in the ribosome, we obtained a cryo-electron microscopy reconstruction of the native large subunit at subnanometer resolution. The insertion site forms a four-way junction that fully preserves the canonical 5S rRNA structure. Moving away from the junction site, the inserted region is conformationally flexible and does not pack tightly against the large subunit.

scholarly journals Structure of the eukaryotic cytoplasmic pre-40S ribosomal subunit

2017 ◽  
Author(s):  
Alain Scaiola ◽  
Cohue Peña ◽  
Melanie Weisser ◽  
Daniel Böhringer ◽  
Marc Leibundgut ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Rna ◽  
Ribosomal Subunit ◽  
Mature Form ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
40S Ribosomal Subunit ◽  
Final Maturation ◽  
Assembly Factors ◽  
Conformational Rearrangements

AbstractFinal maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA. Using cryo-electron microscopy (cryo-EM), we have determined the structure of a cytoplasmic pre-40S particle poised to initiate final maturation at a resolution of 3.4 Å. The structure reveals the extent of conformational rearrangements of the 3’ major and 3’ minor domains of the ribosomal RNA that take place during maturation, as well as the roles of the assembly factors Enp1, Ltv1, Rio2, Tsr1, and Pno1 in the process. Altogether, we provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle towards the mature form capable of engaging in translation.

scholarly journals Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

2018 ◽  
Author(s):  
Dejian Zhou ◽  
Xing Zhu ◽  
Sanduo Zheng ◽  
Dan Tan ◽  
Meng-Qiu Dong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
5.8S Rrna ◽  
Early Processing ◽  
Large Subunit Rrna

AbstractAssembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. How the highly intertwined structure of 60S large ribosomal subunits is established is unknown. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half assembled subunit. Domains I, II and VI of 25S/5.8S rRNA tightly pack into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to the establishment of the global architecture of 60S subunits.

scholarly journals Visualizing formation of the active site in the mitochondrial ribosome

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Viswanathan Chandrasekaran ◽  
Nirupa Desai ◽  
Nicholas O Burton ◽  
Hanting Yang ◽  
Jon Price ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Large Subunit ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Peptidyl Transferase ◽  
Mitochondrial Stress ◽  
Peptidyl Transferase Center ◽  
Cryo Electron Microscopy ◽  
Mitochondrial Ribosome ◽  
Specific Factors

Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2ʹ-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.

scholarly journals The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged Escherichia coli ribosomes

2018 ◽  
Vol 217 (7) ◽  
pp. 2519-2529 ◽  
Author(s):  
Sandip Dey ◽  
Chiranjit Biswas ◽  
Jayati Sengupta
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Ribosomal Rna ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Rna Helicase ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Guanosine Triphosphatase

The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Escherichia coli. Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that E. coli HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo–electron microscopy structure of the 50S–HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival.

scholarly journals Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes

2020 ◽  
Author(s):  
Jennifer N. Wells ◽  
Robert Buschauer ◽  
Timur Mackens-Kiani ◽  
Katharina Best ◽  
Hanna Kratzat ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Stationary Phase ◽  
Large Subunit ◽  
Structure And Function ◽  
Nutrient Deprivation ◽  
Recycling System ◽  
Cryo Electron Microscopy ◽  
And Function

AbstractCells adjust to nutrient deprivation by reversible translational shut down. This is accompanied by maintaining inactive ribosomes in a hibernation state, where they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to Stm1 (SERBP1 in mammals), and recently Lso2 (CCDC124 in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the non-unrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase-activating center of the large subunit. This position allows accommodation of the Dom34-dependent ribosome recycling system, which splits Lso2-containing but not Stm1-containing ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.

scholarly journals Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus

2021 ◽  
Vol 8 ◽  
Author(s):  
Margarita Belinite ◽  
Iskander Khusainov ◽  
Heddy Soufari ◽  
Stefano Marzi ◽  
Pascale Romby ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Structural Biology ◽  
Ribosomal Rna ◽  
Biochemical Characterization ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Dependent Manner ◽  
Positive Bacterium ◽  
Cryo Electron Microscopy

Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 Å and in its absence at 5.3 Å. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.

scholarly journals 4.5S ribonucleic acid, a novel ribosome component in the chloroplasts of flowering plants

1979 ◽  
Vol 183 (3) ◽  
pp. 605-613 ◽  
Author(s):  
C M Bowman ◽  
T A Dyer
Keyword(s):  
Ribosomal Rna ◽  
Broad Bean ◽  
Large Subunit ◽  
Flowering Plants ◽  
5S Rrna ◽  
23S Rrna ◽  
Low Molecular Weight ◽  
5.8S Rrna ◽  
Wide Range ◽  
Predominant Variant

A species of low-molecular-weight ribosomal RNA, referred to as ‘4.5S rRNA’, was found in addition to 5S rRNA in the large subunit of chloroplast ribosomes of a wide range of flowering plants. It was shown by sequence analysis that several variants of this RNA may occur in a plant. Furthermore, although in most flowering plants the predominant variant contains about 100 nucleotides, in the broad bean it has less than 80. It seems, therefore, to be much more diverse in size and sequence than the other ribosomal RNA species. Like 5S rRNA, it does not contain modified nucleotides and it is also unusual in having an unphosphorylated 5′-end. It is apparently neither a homologue of cytosol 5.8S rRNA nor a fragment of 23S rRNA.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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