The 80S rat liver ribosome at 25 å resolution by electron cryomicroscopy and angular reconstitution

Dube, Prakash; Wieske, Martin; Stark, Holger; Schatz, Michael; Stahl, Joachim; Zemlin, F.; Lutsch, Gudrun; Heel, Marin van

doi:10.1016/s0969-2126(98)00040-9

Cited by 53 publications

(44 citation statements)

References 49 publications

(94 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This is in agreement with structural evidence from crystallographic studies, i.e., the ribosomal intersubunit space, including peptidyltransferase and decoding sites, is constructed mainly with conserved rRNA moieties (50,51). Furthermore, cryoelectronmicroscopic studies showed structural resemblance in the interface surfaces of both large and small subunits between rat and E. coli (52). Meanwhile, it is highly likely that there is strong similarity in domains 3, 4, and 5 of the translocases that appear to mimic the structure of tRNA (reviewed in Refs.…”

Section: Figsupporting

confidence: 88%

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

Uchiumi¹,

Honma²,

Nomura³

et al. 2002

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

Ribosomal L10⅐L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0⅐P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1␣ (eEF-1␣) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10⅐L7/L12 complex or L11 with rat protein showed that the P0⅐P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.The "GTPase center" of the ribosome is a region involved in interaction with GTP-bound translation factors, GTP hydrolysis (1), and post-GTPase events including tRNA movements on the ribosome (2, 3). Translation elongation is markedly stimulated by the interaction of this region with two elongation factors in a GTP-dependent manner. The GTPase center includes two essential RNA regions around positions 1070 and 2660 (Escherichia coli numbering) of the 23 S/28 S rRNA (4), which appear to bind to the elongation factors (5, 6). Despite the highly conserved structure of the 1070 and 2660 RNA regions, ribosomes show a kingdom-dependent accessibility for translation factors, i.e., prokaryotic ribosomes do not engage in translation elongation with the eukaryotic factors instead of the prokaryotic factors (7-9). Furthermore, there are differences in the rate of GTPase turnover between the two systems; in vitro eukaryotic eEF-2 1 /80 S ribosomedependent GTP hydrolysis is 10-fold slower than the prokaryotic EF-G/70 S ribosome system (10). This may reflect, in part, the elaborate regulation of eukaryotic translation.The other important component of the GTPase center is the acidic stalk protein, termed L7/L12 in prokaryotes (11-15). Four copies of this proteins bind to protein L10 and form a stable complex (16), designated here as L10⅐L7/L12. This protein complex and another protein, L11, are assembled on the 1070 RNA domain (17). Flexible property of L7/L12 protein in the ribosome (16, 18 -20) seems to be correlated with the fast turnover of EF-G-dependent GTPase. The eukaryotic counterparts of the prokaryotic L7/L12 and L10 are P1/P2 and P0, respectively (21,22)....

show abstract

Section: Figsupporting

confidence: 88%

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

Uchiumi¹,

Honma²,

Nomura³

et al. 2002

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

show abstract

“…The structure of cellular ribonucleoprotein particles has been analyzed by electron microscopy and image processing with great success. By using these techniques, the ribosome has been studied as a single-particle, noncrystalline sample, and progressively improved data has led to three-dimensional reconstructions with a resolution of 15 to 25 Å (13,40). Likewise, the structures of spliceosomal A complex and the large nuclear RNP particle have been determined, although to a lower resolution (17,71).…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural and Functional Analyses of Recombinant Influenza Virus Ribonucleoproteins Suggest Dimerization of Nucleoprotein during Virus Amplification

Ortega

Martín‐Benito

Zürcher

et al. 2000

J Virol

115

130

View full text Add to dashboard Cite

Influenza virus ribonucleoproteins (RNPs) were reconstituted in vivo from cloned cDNAs expressing the three polymerase subunits, the nucleoprotein (NP), and short template RNAs. The structure of purified RNPs was studied by electron microscopy and image processing. Circular and elliptic structures were obtained in which the NP and the polymerase complex could be defined. Comparison of the structure of RNPs of various lengths indicated that each NP monomer interacts with approximately 24 nucleotides. The analysis of the amplification of RNPs with different lengths showed that those with the highest replication efficiency contained an even number of NP monomers, suggesting that the NP is incorporated as dimers into newly synthesized RNPs.The genome of influenza A virus consists of eight ribonucleoprotein complexes (vRNPs) containing a single-stranded RNA segment of negative polarity associated to nucleoprotein (NP) molecules and bound to the polymerase. This enzyme is a heterotrimer formed by the PB1, PB2, and PA proteins (11,12,25,29), all of them being required for efficient RNA transcription and replication (52) (B. Perales, unpublished data). Both transcription and replication take place in the nucleus of the infected cells (24,27). The replication of viral RNA (vRNA) involves the generation of a full-length RNA copy of positive polarity that is encapsidated with NP molecules and complexed with the polymerase (cRNP). These cRNPs serve as intermediates for the synthesis of vRNA progeny molecules (22). For RNA transcription, capped primers generated from cellular hnRNAs by a cap-stealing mechanism (35) are elongated by copying the vRNA template. The termination and polyadenylation signal consists of an oligo(U) sequence located close to the 5Ј terminus of the vRNA templates (59, 64) next to the panhandle structure (38). These processes require the interaction of the polymerase with the conserved 5Ј-terminal sequences of the template (58,61,62).The polymerase domains involved in intersubunit interactions have been identified (21,51,53,73,78), as well as the sequences in PB1 that bind the vRNA template (19, 36) and the cRNA template (20). The PB1 protein contains amino acid motifs present in other RNA-dependent RNA polymerases (56), whose mutation abolishes the transcriptional activity (5). The PB2 subunit is involved in the initiation of viral transcription (3, 51). It is a cap-binding protein (6,70,74) and contains the cap-dependent endonuclease activity (37). The biochemical role of the PA subunit is still uncertain. The phenotypes of temperature-sensitive mutants (reviewed in reference 39) suggest its involvement in vRNA synthesis. The PA subunit is a phosphoprotein (68) whose expression by transfection leads to the degradation of coexpressed proteins (67, 69).The structure of the RNPs present in influenza virions has been studied by electron microscopy (9, 23, 28, 57). They consist of a ribbon-like cord, held together at its ends and folded back to form a coiled structure with a terminal loop. The available e...

show abstract

“…Human ribosomes have the same 80 r proteins that are found in T. aestivum ribosomes and, in terms of rRNA, differ significantly only in the length of four ES on the large subunit (ES7 L , ES15 L , ES27 L , and ES39 L ). These are longer in human (∼850, ∼180, ∼700, and ∼220 nts) than in T. aestivum/yeast (∼200, ∼20, ∼150, and ∼120 nts, respectively), and cryo-EM reconstructions of mammalian ribosomes (23,(42)(43)(44) show that the longer ES in mammalian ribosomes are generally highly mobile elements for which little to no density is visible (Fig. 5).…”

Section: Comparison Of Expansion Segments Between Yeast and Wheat Germmentioning

confidence: 98%

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

Armache

Jarasch

Anger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

203

198

View full text Add to dashboard Cite

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.modeling | molecular dynamics | flexible fitting I n all living cells, the translation of mRNA into polypeptide occurs on ribosomes. Ribosomes provide a platform upon which aminoacyl-tRNAs interact with the mRNA as well as position the aminoacyl-tRNAs for peptide-bond formation (1). Ribosomes are composed of two subunits, a small subunit that monitors the mRNA-tRNA codon-anticodon duplex to ensure fidelity of decoding (2, 3) and a large subunit that contains the active site where peptide-bond formation occurs (4). Both the small and large subunits are composed of RNA and protein: In eubacteria such as Escherichia coli, the small subunit contains one 16S rRNA and 21 ribosomal proteins (r proteins), whereas the large subunit contains 5S and 23S rRNAs and 33 r proteins. Crystal structures of the complete bacterial 70S ribosome were initially reported at 5.5 Å (5), with an interpretation based on atomic models of the individual subunit structures (6-8), and are now available at atomic resolution (9). These structures have provided unparalleled insight into the mechanism of different steps of translation (1) as well as inhibition by antibiotics (10).Compared to the bacterial ribosome, the eukaryotic counterpart is more complicated, containing expansion segments (ES) and variable regions in the rRNA as well as many additional r proteins and r-protein extensions. Plant and fungal 80S ribosomes contain ∼5;500 nucleotides (nts) of rRNA and ∼80 r proteins, whereas bacterial 70S ribosomes comprise ∼4;500 nts and 54 r proteins. The additional elements present in eukaryotic ribosomes may reflect the increased complexity of translation regulation in eukaryotic cells, as evident for assembly, translation initiation, and development, as well as the phenomenon of localized translation (11-15).Early models for eukaryotic ribosomes were derived from electron micrographs of negative-stain or freeze-dried ribosomal particles (16) and localization of r proteins was attempted using immuno-EM and cross-linking approaches; see, for example, refs. 17-20. The first cryo-EM reconstruction of a eukaryotic 80S ribosome was reported for wheat germ (Triticum aestivum) at 38 Å (21). Initial core models for the yeast 80S ribosome were built at 15-Å resolution (22) by docking the rRNA s...

show abstract

The 80S rat liver ribosome at 25 å resolution by electron cryomicroscopy and angular reconstitution

Cited by 53 publications

References 49 publications

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

Ultrastructural and Functional Analyses of Recombinant Influenza Virus Ribonucleoproteins Suggest Dimerization of Nucleoprotein during Virus Amplification

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

Contact Info

Product

Resources

About