scholarly journals A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

2021 ◽  
Vol 7 (23) ◽  
pp. eabf7547
Author(s):  
Andreas Schedlbauer ◽  
Idoia Iturrioz ◽  
Borja Ochoa-Lizarralde ◽  
Tammo Diercks ◽  
Jorge Pedro López-Alonso ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Information ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Structural Description ◽  
Cryo Electron Microscopy ◽  
Bacterial Ribosome ◽  
Core Set ◽  
Assembly Factors ◽  
Conformational Landscape

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo–electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

scholarly journals A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

2020 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Idoia Iturrioz ◽  
Borja Ochoa-Lizarralde ◽  
Tammo Diercks ◽  
Jorge Pedro López-Alonso ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Information ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Structural Description ◽  
Sequential Order ◽  
Bacterial Ribosome ◽  
Core Set ◽  
Assembly Factors ◽  
Conformational Landscape

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria employ an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-EM to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S rRNA central to decoding site maturation, and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

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 Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques

2007 ◽  
Vol 189 (9) ◽  
pp. 3434-3444 ◽  
Author(s):  
M. Jiang ◽  
S. M. Sullivan ◽  
A. K. Walker ◽  
J. R. Strahler ◽  
P. C. Andrews ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Protein Identification ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Absolute Quantitation ◽  
Proteomic Approach ◽  
Assembly Factors ◽  
Associated Proteins ◽  
Isobaric Tags

ABSTRACT Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16°C and 37°C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

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 Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Olga Buzovetsky ◽  
Caitlin Steckler ◽  
Henrik Molina ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Early Maturation ◽  
External Transcribed Spacer ◽  
Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

scholarly journals Structures of substrate complexes of foamy viral protease-reverse transcriptase

2021 ◽  
Author(s):  
Marzena Nowacka ◽  
Elżbieta Nowak ◽  
Mariusz Czarnocki-Cieciura ◽  
Justyna Jackiewicz ◽  
Krzysztof Skowronek ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acid ◽  
Structural Information ◽  
Foamy Virus ◽  
Rnase H ◽  
Full Length ◽  
Structural Description ◽  
Oligomeric State ◽  
Reverse Transcriptases ◽  
Cryo Electron Microscopy

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA, a crucial process for the replication of retroviruses. Foamy viruses (FV) possess a unique RT which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of full-length MFV PR-RT - dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting ssRNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their lifecycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates have been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both monomeric configuration, which is observed in our structures in the presence of RNA/DNA hybrid, and an asymmetric dimer arrangement which we observed in the presence of dsDNA.

scholarly journals Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes

2018 ◽  
Vol 217 (7) ◽  
pp. 2503-2518 ◽  
Author(s):  
Stephanie Biedka ◽  
Jelena Micic ◽  
Daniel Wilson ◽  
Hailey Brown ◽  
Luke Diorio-Toth ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Endonucleolytic Cleavage ◽  
Cryo Electron Microscopy ◽  
External Transcribed Spacer ◽  
Assembly Factors

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo–electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.

scholarly journals Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

2019 ◽  
Author(s):  
Aida Razi ◽  
Joseph H. Davis ◽  
Yumeng Hao ◽  
Dushyant Jahagirdar ◽  
Brett Thurlow ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Small Subunit ◽  
Folding Pathway ◽  
Ribosome Assembly ◽  
Combined Approach ◽  
Quantitative Mass Spectrometry ◽  
Cryo Electron Microscopy ◽  
30S Subunit ◽  
Assembly Factor

SUMMARYTo reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era’s role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

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