Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

Gross, B; Westermann, Peter; Bielka, H

doi:10.1002/j.1460-2075.1983.tb01414.x

Cited by 29 publications

(6 citation statements)

References 20 publications

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“…Additional supporting information was utilized for the localization of r proteins, particular those modeled ab initio. The supporting data included species-specific differences in length between r proteins of wheat germ, yeast, and archaeal ribosomes, as well as the wealth of data available on the spatial arrangement of r proteins in eukaryotic ribosomes derived from a variety of different approaches: (i) the order of assembly of r proteins (29); (ii) accessibility of particular r proteins to proteolysis; (iii) crosslinking of r proteins (15,18,30,31); and (iv) immuno-EM studies (16,32) (see Table S6). Furthermore, the localization of r-protein L38e was supported by comparison of a cryo-EM reconstruction of wild-type yeast 80S ribosome with that of a yeast 80S ribosome isolated from a strain lacking the gene for r-protein L38e ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, the localization of ribosomal proteins within eukaryotic 80S ribosomes has come mainly from early studies using immuno-EM and cross-linking approaches (see, for example, refs. [15][16][17][18]. Moreover, the first molecular models for the eukaryotic ribosome were built at 15-Å resolution by docking the structures of the bacterial small 30S subunit (6) and archaeal large 50S subunit (13), thus only identifying the location of a total of 46 eukaryotic r proteins with bacterial or archaeal homologues (19).…”

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confidence: 99%

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Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Armache

Jarasch

Anger

et al. 2010

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

128

134

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Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74∕80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.homology modeling | RNA | translation | flexible fitting | molecular dynamics P rotein synthesis occurs on large macromolecular complexes, called ribosomes (1). Ribosomes are composed of two subunits, both of which are built from protein and RNA. Bacterial ribosomes, for example, in Escherichia coli, contain a small subunit composed of one 16S rRNA and 21 ribosomal proteins (r proteins), and a large subunit containing 5S and 23S rRNAs and 33 r proteins. In contrast, eukaryotic ribosomes are much larger and more complex, containing additional RNA in the form of so-called expansion segments (ES) as well as many additional r proteins and r-protein extensions. The additional r proteins present in eukaryotic ribosomes are likely to reflect the increased complexity of translation regulation in eukaryotic cells (2-5). Moreover, many of these eukaryote-specific components have been associated with human disorders (4). Thus, structural insight into the localization of these elements will be important to furthering our understanding of eukaryotic translation regulation as well as disease.Compared with the ∼54 r proteins of the bacterial ribosome, plant and fungal 80S ribosomes contain ∼80 r proteins (see Table S1 for r-protein nomenclature). Crystal structures have revealed the location of each small and large subunit r protein within bacterial ribosomes (6-12) as well as the r proteins within the archaeal large ribosomal subunit (13,14). In contrast, the localization of ribosomal proteins within eukaryotic 80S ribosomes has come mainly from early studies using immuno-EM and cross-linking approaches (see, for example, refs. 15-18). Moreover, the first molecular models for the eukaryotic ribosome were built at 15-Å resolution by docking the structures of the bacterial small 30S subunit (6) and archaeal large 50S subunit (13), thus only identifying the location of a total of 46 eukaryotic r proteins with bacterial or archaeal homologues (19). Recently, cryo-EM reconstructions of plant and fungal 80S ribosomes have led to the localization of three eukaryote-specific r proteins: RACK1 (20) and S19e (21) in the small subunit and L30e in the large subunit...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Armache

Jarasch

Anger

et al. 2010

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

128

134

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“…3C). Cross-linking experiments have shown that eIF-3 can be cross-linked to a number of ribosomal proteins depending on the length of the reagent used (31,32). Some of these proteins are clearly interface proteins (14,33), suggesting that part of the binding site for eIF-3 overlaps the subunit interface.…”

Section: Discussionmentioning

confidence: 99%

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Melander

Holmberg

Nygård

1997

Journal of Biological Chemistry

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We have analyzed the structure of 18 S rRNA in native 40 S subunits using chemical modification followed by primer extension. The native subunits were modified using the single-stranded specific reagents dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The modification pattern of the 18 S rRNA was compared to that obtained from 1 binds to 40 S subunits and prevents formation of unprogrammed 80 S ribosomes by inhibiting association of the 40 S and 60 S ribosomal subunits in the absence of mRNA. Initiation factor eIF-2 selects the specific initiator tRNA (MettRNA f ) and brings it to the 40 S subunit. The resulting 43 S pre-initiation complex binds mRNA with the help of a series of initiation factors. The 60 S subunit now joins the mRNA containing 48 S initiation complex in a reaction that requires an additional initiation factor (eIF-5) and is associated with the hydrolysis of GTP.Several of the initiation factors are found to be associated with the so-called native 40 S ribosomal subunits (40 S N ) in vivo (2). Most of these factors are present on the 40 S N particles in small quantities, but eIF-3 is present in stoichiometric amounts (2). Initiation factor 3 is a huge multisubunit protein with a total mass of approximately 0.7 MDa (3). The factor displays RNA binding properties, and one of its subunits can be cross-linked to 18 S rRNA in the 40 S⅐eIF-3 complex (4). This suggests that rRNA may, at least in part, be responsible for binding the factor to the small ribosomal subunit. However, the location of the eIF-3 interaction site in 18 S rRNA is not known.The ribosomal RNA is considered to be involved in various ribosomal functions such as A-and P-site-related activities and peptide bond formation (for a review see Ref. 5). In prokaryotes the rRNA is directly involved in the binding of initiation factors and mRNA during protein synthesis initiation (6 -9). Less is known about the functional role of rRNA in the eukaryotic ribosome, but studies using chemical cross-linking and chemical and enzymatic footprinting have indicated that the rRNA is involved in mRNA binding, subunit interaction, and binding of elongation factors (10 -12).We have previously studied the structure of 18 S rRNA in derived 40 S subunits prepared by dissociation of isolated 80 S ribosomes (11, 13). In contrast to the native subunits, derived particles are free from additional non-ribosomal proteins. In this report, we have compared the structures of 18 S rRNA in native and derived 40 S subunits using chemical modification. The two types of 18 S rRNAs showed distinct but limited structural differences. The role of the non-ribosomal proteins in altering the structure of the 18 S rRNA in the 40 S N particles is discussed. Nygård and Nika (14). Briefly, isolated monosomes (15) were suspended in 0.5 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl 2 , and 10 mM 2-mercaptoethanol. The material was layered onto continuous 10 -40% (w/v) sucrose gradients containing 0.35 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl ...

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“…Chemical cross-linking studies have provided much information on protein neighborhoods in mammalian ribosomal small [6,[19][20][21] and large subunits [4, 5, 221. In this study we applied the cross-linking method to localize the binding sites for EF-2 on rat liver 80s ribosomes, employing 2-iminothiolane.…”

Section: Discussionmentioning

confidence: 99%

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

et al. 1986

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Complexes containing rat liver 80s ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[/3,y-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with lZ5I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of M, 33000), P2, S6 and S23/ S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33 -L11 and LA33 -L12.During protein biosynthesis in eukaryotic cells, elongation factor 2 (EF-2) interacts with ribosomes in the presence of GTP and catalyzes the translocation of newly synthesized peptidyl-tRNA from the aminoacyl-tRNA binding site (the A site) of ribosomes to the peptidyl-tRNA site (the P site) [l -31. The functional roles of ribosomal proteins in the translocation are as yet unknown. To understand the molecular mechanisms of the translocation in mammalian ribosomes, it is important to identify proteins in the EF-2 binding site of ribosomes.Employing four kinds of cross-linking reagents and hydrogen peroxide, we identified 62 cross-linked protein pairs involving 36 protein species containing two acidic proteins of rat liver 60s subunits and have discussed these in relation to other functional data [4, 51. Concerning the 40s subunits, similar data were presented by Tolan and Traut [6]. Therefore, it is of interest that ribosomal proteins which interact with elongation factors have been determined.In the present experiments we investigated EF-2-binding sites in rat liver 80s ribosomes, employing 2-iminothiolane. Cross-linked proteins were analyzed by a modified method of diagonal SDS gel electrophoresis [7]. Seven large-subunit proteins and two small-subunit proteins were identified as the components cross-linked to EF-2. The mutual locations of these proteins in the 80s ribosomes are discussed.

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Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

Cited by 29 publications

References 20 publications

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

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