Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit

Ban, Nenad; Nissen, Poul; Hansen, J. Lundsgaard; Capel, Malcolm; Moore, Peter B.; Steitz, Thomas A.

doi:10.1038/23641

Cited by 390 publications

(285 citation statements)

References 45 publications

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“…The combination of these effects would suggest that it is likely to be sensitive to perturbations in protein-RNA interactions. Similarly, the binding site of L6 is established in close proximity to the factor binding site (34) (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

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Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Hanson¹,

Fucini²,

Ilag³

et al. 2003

Journal of Biological Chemistry

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We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg 2؉ by Li ؉ ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.

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Section: Resultsmentioning

confidence: 99%

“…1. The structure of EF-G (Protein Data Bank code 1FNM) was fitted according to the structure of Ban et al (34). The proteins colored in the two structures are those released from the two complexes.…”

Section: Resultsmentioning

confidence: 99%

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Hanson¹,

Fucini²,

Ilag³

et al. 2003

Journal of Biological Chemistry

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“…Similarly, models for 44 of the 80 r proteins of the T. aestivum 80S ribosome (gray in Fig. 2 E and F) were built using the templates present in the bacterial and archaeal ribosome structures (29,30), as well as 44 of 79 r proteins of the yeast 80S ribosome (see ref. 26).…”

Section: Resultsmentioning

confidence: 99%

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.

198

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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...

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“…P0, contrary to L10, contains the flexible alanine-rich region and the phosphorylable C-terminal domain found in P1 and P2. Mobility complicates the study of the components of the stalk that represent two of the last three proteins not shown in the crystallographic structure of the 50 S ribosomal subunit of the archaea, Haloarcula marismortui (25,26). Besides, structural studies of the isolated proteins are incomplete (27).…”

mentioning

confidence: 99%

Pivotal Role of the P1 N-terminal Domain in the Assembly of the Mammalian Ribosomal Stalk and in the Proteosynthetic Activity

Gonzalo

Lavergne

Reboud

2001

Journal of Biological Chemistry

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In the 60 S ribosomal subunit, the lateral stalk made of the P-proteins plays a major role in translation. It contains P0, an insoluble protein anchoring P1 and P2 to the ribosome. Here, rat recombinant P0 was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. This method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with P0. Furthermore, the use of truncated mutants of P1 and P2 indicated that residues 1-63 in P1 connected P0 to residues 1-65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with P0 or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant P0 in complex with phosphorylated P2 and either P1 or its (1-63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded P0 was functional and residues 1-63 only in P1 were essential. Furthermore, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.The ribosome is the central constituent of the protein synthesis machinery (1). During translation of messenger RNA into protein, the ribosome is helped by several soluble factors that operate in a sequential manner to improve both efficiency and fidelity of this process (2, 3). How this extraordinary coordination is performed is not yet exactly understood. Still, because most of the translation factors are GTPases, the driving of the factors by the ribosome is likely to be controlled by GTP hydrolysis (4). A small portion of the 28 S rRNA designated as the GTPase center is known to be involved in GTP hydrolysis activation (5). The GTPase center is connected directly to the stalk (6), an elongated and very flexible protuberance interacting with elongation factors (7-10). However, despite decades of research, the organization and functions of the proteins constituting the stalk remain unclear. The number and the nature of the proteins constituting the stalk are different depending on the biological system, although their general organization, made of five proteins, is likely to be similar. In prokaryotes, four identical proteins (L7/L12) are linked to the GTPase center by L10 connected itself with a sixth protein, L11 (11). In mammals, the equivalents of the four L7/L12s are two different proteins, P1 and P2, each being present in two copies. These proteins are bound to P0, the equivalent of L10, which is itself bound to L12, the eukaryotic equivalent of L11, and to the GTPase center (6, 12). In plants, an additional protein, P3, has been described (13). In yeast, there are two variants of both P1 (P1␣ and P1␤) and P2 (P2␣ and P2␤); the precise repartition and function of each variant remains unsettled (14). This structural heterogeneity seems to correspond to functional differences, and data obtained in one system cannot be extrapolated directly (10). Bot...

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Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit

Cited by 390 publications

References 45 publications

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

Dissociation of Intact Escherichia coli Ribosomes in a Mass Spectrometer

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

Pivotal Role of the P1 N-terminal Domain in the Assembly of the Mammalian Ribosomal Stalk and in the Proteosynthetic Activity

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