scholarly journals Ribosome assembly factors modulate the RNA folding landscape to prevent misfolding

2020 ◽  
Author(s):  
Haina Huang ◽  
Katrin Karbstein
Keyword(s):  
Structure Analysis ◽  
Rna Folding ◽  
Ribosomal Subunit ◽  
Folding Pathway ◽  
Ribosome Assembly ◽  
Yeast Genetics ◽  
Native Structure ◽  
Interaction Partners ◽  
Assembly Factors

AbstractRibosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head. Our data demonstrate how a succession of assembly factors prevent the early folding of an RNA loop, thereby allowing an adjacent helical junction to fold first. Deletion of the assembly factors to allow the loop to fold first traps a misfolded junction, demonstrating how assembly factors modulate the folding pathway to enable challenging motifs to fold independent of their interaction partners and guide folding to the native structure. Analysis of ribosome intermediate structures indicates that this role for assembly factors is ubiquitous and suggests that helical junctions present significant obstacles to folding.

scholarly journals Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

Abstract Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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 Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

AbstractFolding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

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 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 The Small-Subunit Processome Is a Ribosome Assembly Intermediate

2004 ◽  
Vol 3 (6) ◽  
pp. 1619-1626 ◽  
Author(s):  
Kara A. Bernstein ◽  
Jennifer E. G. Gallagher ◽  
Brianna M. Mitchell ◽  
Sander Granneman ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Bona Fide ◽  
Protein Components ◽  
Ssu Processome

ABSTRACT The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5′ end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

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 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.

scholarly journals The p21-Activated Protein Kinase Inhibitor Skb15 and Its Budding Yeast Homologue Are 60S Ribosome Assembly Factors

2007 ◽  
Vol 27 (8) ◽  
pp. 2897-2909 ◽  
Author(s):  
Cosmin Saveanu ◽  
Jean-Claude Rousselle ◽  
Pascal Lenormand ◽  
Abdelkader Namane ◽  
Alain Jacquier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ribosome Biogenesis ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Budding Yeast ◽  
Ribosomal Subunit ◽  
Yeast Cells ◽  
Ribosome Assembly ◽  
Main Function

ABSTRACT Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G1, and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.

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