Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

Abstract The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

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Assembling the archaeal ribosome: roles for translation-factor-related GTPases

Biochemical Society Transactions ◽

10.1042/bst0390045 ◽

2011 ◽

Vol 39 (1) ◽

pp. 45-50 ◽

Cited By ~ 12

Author(s):

Fabian Blombach ◽

Stan J.J. Brouns ◽

John van der Oost

Keyword(s):

Conformational Changes ◽

Ribosomal Proteins ◽

Major Type ◽

Ribosome Assembly ◽

Ribosomal Subunits ◽

Translation Factor ◽

Associated Functions ◽

Assembly Factor ◽

Switch Mechanism ◽

Stepwise Assembly

The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a ‘molecular switch’ mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo–eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.

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Modular assembly of the nucleolar large subunit processome

10.1101/223412 ◽

2017 ◽

Cited By ~ 1

Author(s):

Zahra Assur Sanghai ◽

Linamarie Miller ◽

Kelly R. Molloy ◽

Jonas Barandun ◽

Mirjam Hunziker ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Molecular Mimicry ◽

Large Subunit ◽

Ribosome Assembly ◽

Modular Assembly ◽

Assembly Result ◽

Concerted Activity ◽

Folding Pathways ◽

Exit Tunnel

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 Ångstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

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Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621129114 ◽

2017 ◽

Vol 114 (6) ◽

pp. 1305-1310 ◽

Cited By ~ 88

Author(s):

Marc Wehmer ◽

Till Rudack ◽

Florian Beck ◽

Antje Aufderheide ◽

Günter Pfeifer ◽

...

Keyword(s):

Atp Hydrolysis ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Core Particle ◽

Nucleotide Analogs ◽

The Core ◽

Cryo Electron Microscopy ◽

Intracellular Proteins ◽

Ubiquitin Proteasome ◽

Structural Insights

In eukaryotic cells, the ubiquitin–proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

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Quantitative Mining of Compositional Heterogeneity in Cryo-EM Datasets of Ribosome Assembly Intermediates

10.1101/2021.06.23.449614 ◽

2021 ◽

Author(s):

Jessica N Rabuck-Gibbons ◽

Dmitry Lyumkis ◽

James R Williamson

Keyword(s):

Structural Heterogeneity ◽

Ribosome Assembly ◽

Compositional Heterogeneity ◽

Assembly Process ◽

Branch Points ◽

Macromolecular Complexes ◽

E Coli ◽

Cryo Electron Microscopy ◽

Assembly Factor ◽

Structural Configurations

Macromolecular complexes are dynamic entities whose function is often intertwined with their many structural configurations. Single particle cryo-electron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct populations into different groups through computational classification. However, current workflows are limited, and there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for a set of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which is characterized by significant structural heterogeneity. We identified new branch points in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

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Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms20112806 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2806 ◽

Cited By ~ 2

Author(s):

Jesse M. Fox ◽

Rebekah L. Rashford ◽

Lasse Lindahl

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Assembly ◽

Relative Timing ◽

Ribosomal Subunits ◽

Eukaryotic Ribosome ◽

Rrna Molecule ◽

Ribosomal Precursor ◽

Its1 And Its2

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.

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

Science ◽

10.1126/science.abb4119 ◽

2020 ◽

Vol 369 (6510) ◽

pp. 1470-1476 ◽

Cited By ~ 5

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.

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Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation–Contraction Complex

Annual Review of Genetics ◽

10.1146/annurev-genet-120417-031311 ◽

2018 ◽

Vol 52 (1) ◽

pp. 373-396 ◽

Cited By ~ 12

Author(s):

Raika Pancaroglu ◽

Filip Van Petegem

Keyword(s):

Ion Channels ◽

Action Potentials ◽

Cryo Electron Microscopy ◽

New Information ◽

Secondary Messengers ◽

Genes Encoding ◽

Associated Proteins ◽

Multiple Structures ◽

Structural Insights ◽

Channel Structures

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

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

10.1101/525360 ◽

2019 ◽

Cited By ~ 1

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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Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

10.1101/2021.02.02.429294 ◽

2021 ◽

Author(s):

Yuhei Chadani ◽

Nobuyuki Sugata ◽

Tatsuya Niwa ◽

Yosuke Ito ◽

Shintaro Iwasaki ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Amino Acid Residues ◽

Translation Elongation ◽

Ribosomal Subunits ◽

Nascent Polypeptide ◽

Nascent Chain ◽

Exit Tunnel ◽

Living Organisms

SummaryContinuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

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Symmetry at the active site of the ribosome: structural and functional implications

Biological Chemistry ◽

10.1515/bc.2005.098 ◽

2005 ◽

Vol 386 (9) ◽

pp. 833-844 ◽

Cited By ~ 71

Author(s):

Ilana Agmon ◽

Anat Bashan ◽

Raz Zarivach ◽

Ada Yonath

Keyword(s):

Ribosomal Rna ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Entire Region ◽

Peptidyl Transferase ◽

Ribosomal Subunits ◽

Peptidyl Transferase Center ◽

Functional Implications ◽

Exit Tunnel ◽

Tunnel Entrance

Abstract The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3′-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3′-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2′-hydroxyl.

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