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

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.

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Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

10.1101/2021.05.18.444632 ◽

2021 ◽

Author(s):

Philipp Milkereit ◽

Gisela Pöll ◽

Michael Pilsl ◽

Joachim Griesenbeck ◽

Herbert Tschochner

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Protein Assembly ◽

Functional Coupling ◽

Large Ribosomal Subunit ◽

Cryo Electron Microscopy ◽

Intrinsic Quality ◽

Domain Arrangement ◽

The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Molecules ◽

10.3390/molecules25051125 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1125 ◽

Cited By ~ 3

Author(s):

Ramtin Shayan ◽

Dana Rinaldi ◽

Natacha Larburu ◽

Laura Plassart ◽

Stéphanie Balor ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Particle Analysis ◽

Ribosomal Subunits ◽

Early Maturation ◽

Cryo Electron Microscopy ◽

Molecular Events ◽

Final Maturation

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

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Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors

PLoS ONE ◽

10.1371/journal.pone.0252497 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0252497

Author(s):

Gisela Pöll ◽

Michael Pilsl ◽

Joachim Griesenbeck ◽

Herbert Tschochner ◽

Philipp Milkereit

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Protein Assembly ◽

Functional Coupling ◽

Large Ribosomal Subunit ◽

Cryo Electron Microscopy ◽

Intrinsic Quality ◽

Domain Arrangement ◽

The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit

Nucleic Acids Research ◽

10.1093/nar/gkz1079 ◽

2019 ◽

Cited By ~ 1

Author(s):

Christina M Braun ◽

Philipp Hackert ◽

Catharina E Schmid ◽

Markus T Bohnsack ◽

Katherine E Bohnsack ◽

...

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Small Subunit ◽

Large Ribosomal Subunit ◽

Rrna Sequence ◽

Ribosomal Subunits ◽

External Transcribed Spacer ◽

Exit Tunnel ◽

Domain Iii

Abstract More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The stepwise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a hierarchical manner to ensure correct maturation of the pre-rRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5′ external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5′ ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of ribosomal subunits.

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The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly

Journal of Bacteriology ◽

10.1128/jb.00444-06 ◽

2006 ◽

Vol 188 (19) ◽

pp. 6757-6770 ◽

Cited By ~ 98

Author(s):

Mengxi Jiang ◽

Kaustuv Datta ◽

Angela Walker ◽

John Strahler ◽

Pia Bagamasbad ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Large Subunit ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Macromolecular Complex ◽

Large Ribosomal Subunit ◽

Proteomic Approach ◽

Temporal Relationships

ABSTRACT The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

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A Possible Role for 5 S rRNA as a Bridge Between Ribosomal Subunits

Canadian Journal of Biochemistry ◽

10.1139/o73-224 ◽

1973 ◽

Vol 51 (12) ◽

pp. 1669-1672 ◽

Cited By ~ 23

Author(s):

A. A. Azad ◽

B. G. Lane

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Large Subunit ◽

Ribosomal Subunit ◽

Small Subunit ◽

Low Molecular Weight ◽

Large Ribosomal Subunit ◽

Ribosomal Subunits ◽

Wheat Embryo ◽

Present Context

18 S rRNA is a high molecular weight polyribonucleotide found in the small subunit, and 26 S rRNA is a high molecular weight polyribonucleotide found in the large subunit, whereas 5 S rRNA and 5.8 S rRNA are low molecular weight ("satellite") polyribonucleotides confined to the large subunit of wheat-embryo ribosomes. Under the same conditions in which 5.8 S rRNA is known to complex efficiently and preferentially with 26 S rRNA, it has been observed that 5 S rRNA complexes efficiently and preferentially with 18 S rRNA. Since 5 S rRNA is a component of the large ribosomal subunit, but it complexes preferentially with 18 S rRNA, which is a component of the small ribosomal subunit, it has been proposed that 5 S rRNA may serve as a "bridge" to mediate reversible association between the small and large ribosomal subunits. The possible role that a polycistronic precursor of rRNA might be visualized to play in the biogenesis and assembly of reversibly associating ribosomal subunits is alluded to in the present context.

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Alternative Conformations and Motions Adopted by 30S Ribosomal Subunits Visualized by Cryo-Electron Microscopy

10.1101/2020.03.21.001677 ◽

2020 ◽

Cited By ~ 2

Author(s):

Dushyant Jahagirdar ◽

Vikash Jha ◽

Kaustuv Basu ◽

Josue Gomez-Blanco ◽

Javier Vargas ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

16S Rrna ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Conformational Space ◽

Inactive Conformation ◽

Cryo Electron Microscopy ◽

30S Subunit

ABSTRACTIt is only after recent advances in cryo-electron microscopy that is now possible to describe at high resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between the “active” and “inactive” conformations. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6Å resolution and study its motions. In the inactive conformation, three nucleotides at the 3’ end of the 16S rRNA cause the region of helix 44 forming the decoding center to adopt an unlatched conformation and the 3’ end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42 °C reverts these structural changes. The position adopted by helix 44 dictates the most prominent motions of the 30S subunit. We found that extended exposures to low magnesium concentrations induces unfolding of large rRNA structural domains. The air-water interface to which ribosome subuints are exposed during sample preparation also peel off some ribosomal proteins. Overall this study provides new insights about the conformational space explored by the 30S ribosomal subunit when the ribosomal particles are free in solution.

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Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

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