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

2019 ◽  
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.

scholarly journals Structural Insight into the Antibiotic Action of Telithromycin against Resistant Mutants

2003 ◽  
Vol 185 (14) ◽  
pp. 4276-4279 ◽  
Author(s):  
Rita Berisio ◽  
Joerg Harms ◽  
Frank Schluenzen ◽  
Raz Zarivach ◽  
Harly A. S. Hansen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Deinococcus Radiodurans ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Resistant Strains ◽  
Structural Insight ◽  
Resistant Mutants ◽  
Exit Tunnel ◽  
Structural Insights ◽  
Insight Into

ABSTRACT The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.

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.

scholarly journals Multiple GTPases Participate in the Assembly of the Large Ribosomal Subunit in Bacillus subtilis

2006 ◽  
Vol 188 (23) ◽  
pp. 8252-8258 ◽  
Author(s):  
Laura Schaefer ◽  
William C. Uicker ◽  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Jean-Michel Jault ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Protein Composition ◽  
Large Ribosomal Subunit ◽  
Sucrose Density Gradient Centrifugation ◽  
Direct Role

ABSTRACT GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.

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

2006 ◽  
Vol 188 (19) ◽  
pp. 6757-6770 ◽  
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.

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 The Archaeal Elongation Factor EF-2 Induces the Release of aIF6 From 50S Ribosomal Subunit

2021 ◽  
Vol 12 ◽  
Author(s):  
Giada Lo Gullo ◽  
Maria Luisa De Santis ◽  
Alessandro Paiardini ◽  
Serena Rosignoli ◽  
Alice Romagnoli ◽  
...  
Keyword(s):  
Large Subunit ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Structural Data ◽  
Small Subunit ◽  
Large Ribosomal Subunit ◽  
Homologous Proteins ◽  
Gtp Hydrolysis ◽  
Elongation Factor 2 ◽  
Docking Protein

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.

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

1973 ◽  
Vol 51 (12) ◽  
pp. 1669-1672 ◽  
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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