90S pre-ribosome transformation into the primordial 40S subunit

Science ◽  
2020 ◽  
Vol 369 (6510) ◽  
pp. 1470-1476 ◽  
Author(s):  
Jingdong Cheng ◽  
Benjamin Lau ◽  
Giuseppe La Venuta ◽  
Michael Ameismeier ◽  
Otto Berninghausen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Helicase ◽  
Rna Cleavage ◽  
En Bloc ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
External Transcribed Spacer ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Shed Light

Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. We elucidate this process by biochemical and cryo–electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by Utp24 endonuclease–driven RNA cleavage at site A1, thereby separating the 5′-external transcribed spacer (ETS) from 18S ribosomal RNA. Next, the 5′-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to unwind the final small nucleolar U3–18S RNA hybrid. Our data shed light on the elusive 90S to pre-40S transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.

scholarly journals Isolation of Disease-relevant p53 for Cryo-Electron Microscopy Analysis

2021 ◽  
Author(s):  
Maria J. Solares ◽  
GM Jonaid ◽  
William Y. Luqiu ◽  
Yanping Liang ◽  
Madison C. Evans ◽  
...  
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
Tp53 Gene ◽  
Tumor Suppressor Protein ◽  
Protein Assemblies ◽  
Cryo Electron Microscopy ◽  
Naturally Occurring ◽  
Imaging Protocols ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Abstract Tumor suppressor protein TP53 (p53) plays a multi-faceted role in all cells of thehuman body. Sadly, mutations in the TP53 gene are involved in nearly ~50% of tumors,spurring erratic cell growth and disease progression. Until recently, structural informationfor p53 remained incomplete and there are limited studies on native p53 produced inhuman tumors. Here, we present a highly reproducible and effective protocol to extract,enrich, and purify native p53 protein assemblies from cancer cells for downstreamstructural studies. This method does not introduce purification tags into the p53 gene andmaintains naturally occurring modifications. In conjunction with cryo-Electron Microscopytechniques, we determined new structures for p53 monomers (~50 kDa) and tetramers(~200 kDa) at spatial resolutions of ~4.8 Å and ~7 Å, respectively.1 These modelsrevealed new insights for flexible regions of p53 along with biologically-relevantubiquitination sites. Combining biochemical and structural imaging protocols, we aim tobuild a better understanding of native p53’s impact in cancer formation.

Eukaryotic Ribosome Assembly

2019 ◽  
Vol 88 (1) ◽  
pp. 281-306 ◽  
Author(s):  
Jochen Baßler ◽  
Ed Hurt
Keyword(s):  
Ribosomal Rna ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Cryo Electron Microscopy ◽  
A Cell ◽  
Cellular Pathways ◽  
Nucleolar Rnas ◽  
Shed Light

Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo–electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.

scholarly journals Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

2020 ◽  
Author(s):  
Dongchun Ni ◽  
Kelvin Lau ◽  
Frank Lehmann ◽  
Andri Fränkl ◽  
David Hacker ◽  
...  
Keyword(s):  
Structural Rearrangement ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Angiotensin Converting Enzyme 2 ◽  
Cryo Electron Microscopy ◽  
Monomeric Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Viral Surface

AbstractThe human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

scholarly journals Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex

Science ◽  
2020 ◽  
Vol 368 (6489) ◽  
pp. eaaz2449 ◽  
Author(s):  
Xudong Wu ◽  
Marc Siggel ◽  
Sergey Ovchinnikov ◽  
Wei Mi ◽  
Vladimir Svetlov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ubiquitin Ligase ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Ubiquitin Ligase Complex ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Simulation Results ◽  
Membrane Region

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo–electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two “half-channels” with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

scholarly journals Molecular architecture of the 90S small subunit pre-ribosome

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Qi Sun ◽  
Xing Zhu ◽  
Jia Qi ◽  
Weidong An ◽  
Pengfei Lan ◽  
...  
Keyword(s):  
Small Subunit ◽  
Molecular Architecture ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
U3 Snorna ◽  
Density Maps ◽  
External Transcribed Spacer ◽  
Assembly Factors ◽  
Protein Components ◽  
Insight Into

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

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.

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