scholarly journals Tightly-orchestrated rearrangements govern catalytic center assembly of the ribosome

2018 ◽  
Author(s):  
Yi Zhou ◽  
Sharmishtha Musalgaonkar ◽  
Arlen W. Johnson ◽  
David W. Taylor
Keyword(s):  
Catalytic Activity ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Critical Element ◽  
Large Ribosomal Subunit ◽  
Peptidyl Transferase ◽  
Catalytic Center ◽  
Peptidyl Transferase Center ◽  
Final Assembly ◽  
Cytoplasmic Maturation

AbstractThe catalytic activity of the ribosome is mediated by RNA, yet proteins are essential for the function of the peptidyl transferase center (PTC). In eukaryotes, final assembly of the PTC occurs in the cytoplasm by insertion of the ribosomal protein Rpl10. We determine structures of six intermediates in late nuclear and cytoplasmic maturation of the large subunit that reveal a tightly-choreographed sequence of protein and RNA rearrangements controlling the insertion of Rpl10. We also determine the structure of the biogenesis factor Yvh1 and show how it promotes assembly of the P stalk, a critical element for recruitment of GTPases that drive translation. Together, our structures provide a blueprint for final assembly of a functional ribosome.One Sentence SummaryCryo-EM structures of six novel intermediates in the assembly of the large ribosomal subunit reveal mechanism of creating the catalytic center.

scholarly journals Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariam Jaafar ◽  
Julia Contreras ◽  
Carine Dominique ◽  
Sara Martín-Villanueva ◽  
Régine Capeyrou ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rna Helicase ◽  
Genetic Interactions ◽  
Large Ribosomal Subunit ◽  
Base Pairing ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center

AbstractSynthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

scholarly journals MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

2014 ◽  
Vol 25 (17) ◽  
pp. 2542-2555 ◽  
Author(s):  
Joanna Rorbach ◽  
Pierre Boesch ◽  
Payam A. Gammage ◽  
Thomas J. J. Nicholls ◽  
Sarah F. Pearce ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rna Processing ◽  
Large Subunit ◽  
Peptidyl Transferase ◽  
Mitochondrial Translation ◽  
Peptidyl Transferase Center ◽  
Mitochondrial Ribosome ◽  
Translation Apparatus ◽  
Rrna Maturation ◽  
And Function

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.

scholarly journals Initiation factor 2 stabilizes the ribosome in a semirotated conformation

2015 ◽  
Vol 112 (52) ◽  
pp. 15874-15879 ◽  
Author(s):  
Clarence Ling ◽  
Dmitri N. Ermolenko
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Bound State ◽  
Initiation Factor ◽  
Large Ribosomal Subunit ◽  
Control Of Gene Expression ◽  
Initiation Phase ◽  
Intersubunit Rotation

Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNAfMet, and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.

scholarly journals The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis

2020 ◽  
Vol 48 (21) ◽  
pp. 12310-12325
Author(s):  
Caroline Lacoux ◽  
Ludivine Wacheul ◽  
Kritika Saraf ◽  
Nicolas Pythoud ◽  
Emmeline Huvelle ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Ribosome Biogenesis ◽  
Translation Termination ◽  
Ribosomal Subunit ◽  
Efficient Production ◽  
Large Ribosomal Subunit ◽  
Termination Factor ◽  
Domains Of Life ◽  
Quality Control Mechanism ◽  
Translation Termination Factor

Abstract The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.

scholarly journals Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center

2006 ◽  
Vol 50 (4) ◽  
pp. 1458-1462 ◽  
Author(s):  
Katherine S. Long ◽  
Lykke H. Hansen ◽  
Lene Jakobsen ◽  
Birte Vester
Keyword(s):  
Drug Targets ◽  
Rational Design ◽  
Ribosomal Subunit ◽  
Binding Pocket ◽  
Chain Extension ◽  
Side Chain ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Binding Surface ◽  
The Common

ABSTRACT Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

scholarly journals The Structure of the Yeast Mitochondrial Large Ribosomal Subunit

2014 ◽  
Vol 70 (a1) ◽  
pp. C1051-C1051
Author(s):  
Alan Brown ◽  
Alexey Amunts ◽  
Xiao-chen Bai ◽  
Jose Llácer ◽  
Tanweer Hussain ◽  
...  
Keyword(s):  
Single Particle ◽  
De Novo ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Atomic Model ◽  
Docking Site ◽  
Large Ribosomal Subunit ◽  
Exit Tunnel

Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle electron cryo-microscopy. The resolution of 3.2 Ångstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site and a putative membrane docking site.

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

2018 ◽  
Author(s):  
Dejian Zhou ◽  
Xing Zhu ◽  
Sanduo Zheng ◽  
Dan Tan ◽  
Meng-Qiu Dong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
5.8S Rrna ◽  
Early Processing ◽  
Large Subunit Rrna

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.

scholarly journals Species Specificity in Ribosome Biogenesis: a Nonconserved Phosphoprotein Is Required for Formation of the Large Ribosomal Subunit in Trypanosoma brucei

2005 ◽  
Vol 4 (1) ◽  
pp. 30-35 ◽  
Author(s):  
Bryan C. Jensen ◽  
Deirdre L. Brekken ◽  
Amber C. Randall ◽  
Charles T. Kifer ◽  
Marilyn Parsons
Keyword(s):  
Trypanosoma Brucei ◽  
Small Rnas ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Protozoan Parasite ◽  
Growth Defect ◽  
Large Ribosomal Subunit ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT In the protozoan parasite Trypanosoma brucei, the large rRNA, which is a single 3.4- to 5-kb species in most organisms, is further processed to form six distinct RNAs, two larger than 1 kb (LSU1 and LSU2) and four smaller than 220 bp. The small rRNA SR1 separates the two large RNAs, while the remaining small RNAs are clustered at the 3′ end of the precursor rRNA. One would predict that T. brucei possesses specific components to carry out these added processing events. We show here that the trypanosomatid-specific nucleolar phosphoprotein NOPP44/46 is involved in this further processing. Cells depleted of NOPP44/46 by RNA interference had a severe growth defect and demonstrated a defect in large-ribosomal-subunit biogenesis. Concurrent with this defect, a significant decrease in processing intermediates, particularly for SR1, was seen. In addition, we saw an accumulation of aberrant processing intermediates caused by cleavage within either LSU1 or LSU2. Though it is required for large-subunit biogenesis, we show that NOPP44/46 is not incorporated into the nascent particle. Thus, NOPP44/46 is an unusual protein in that it is both nonconserved and required for ribosome biogenesis.

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