Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3.small ribosomal subunit, as shown by cross-linking experiments

Nygård, Odd; Westermann, Peter

doi:10.1093/nar/10.4.1327

Cited by 45 publications

(29 citation statements)

References 18 publications

(5 reference statements)

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“…As we mentioned above, a group of factors, L1-L4, are linked to 28s RNA. A similar specific interaction was found in rat liver between initiation factor eIF-3 and 18s RNA [4].…”

Section: Discussionsupporting

confidence: 77%

“…During the last few years, covalent cross-linking techniques have been introduced for investigating the structural organisation of the ribosomes. Chemical reagents have been used in order to identify the binding sites for initiation factors [3,4] and to obtain information on the protein topography of both subunits [5 -71. Ultraviolet irradiation has been found to produce RNA-protein cross-links [8].…”

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confidence: 99%

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Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells

Cazillis

Giocanti

Houssais

et al. 1984

European Journal of Biochemistry

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The 40s and 60s ribosomal subunits of L cells are y-irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross-links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups.We found that about 15 proteins are cross-linked to the 28s RNA. Most of them belong to the core proteins of the 60s ribosomal subunits. A few high-molecular mass proteins which might be factors are also bound to 28s RNA.Between 8 and 11 proteins are covalently linked to 18s RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit.The contribution of these results to the study of the three-dimensional ribosomal structure is also discussed.The proteins and nucleic acids in ribosomes are arranged in a specific three-dimensional configuration to form structural and functional domains. Several studies have been made to identify the proteins that interact with the different species of MgSO,. Buffer I11 : 10 mM phosphate buffer pH 7.4, 0.5 mM MgSO,, 200 mM LiCl, 0.1 % sodium dodecylsulfate (SDS).Buffer IV: 10mM phosphate buffer pH 7.4, 100mM LiC1. rRNA~in eukaryotes [l, 21. During the last few years, covalent cross-linking techniques have been introduced for investigating the structural organisation of the ribosomes. Chemical reagents have been used in order to identify the binding sites for initiation factors [3,4] and to obtain information on the protein topography of both subunits [5 -71. Ultraviolet irradiation has been found to produce RNA-protein cross-links [8]. All these data are only now being integrated in terms of the ribosomal three-dimensional structure. Recently, y radiations appeared as an original and valuable tool to induce highly specific RNAprotein covalent bonds in Escherichia coli ribosomes [9,10]. At low doses, they reach the reactive targets in situ, while preserving the structure. Only a few chemical groups are capable of forming radicals and these reactive groups must be very close to form a covalent link [9, lo]. In order to gain more insight on the eukaryotic ribosomal structure, we applied, in this study, the y radiation technique to 40s and 60s ribosomal subunits of L cells. The proteins found cross-linked to rRNA have been identified among the distinct classes of ribosomal proteins defined in a previous study: those split off by KC1, core, transferable and labelled proteins [l 11.These results are discussed in a topological approach to the relationship of the RNA and the protein in the structure of the two ribosomal subunits. MATERIALS AND METHODS BuffersBuffer I contains 10 mM phosphate buffer pH 7.4, 5 mM MgSO,. Buffer I1 : 10 mM phosphate buffer pH 7.4, 0.5 mM Abbreviation. SDS, sodium dodecyl sulfate. Cell growth and labelling conditionsMouse C1 I D LM (TK-) cells were grown in suspension in an Eagle-Dulbecco medium supplemented with 10 % calf serum [12]. For la...

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“…As we mentioned above, a group of factors, L1-L4, are linked to 28s RNA. A similar specific interaction was found in rat liver between initiation factor eIF-3 and 18s RNA [4].…”

Section: Discussionsupporting

confidence: 77%

mentioning

confidence: 99%

Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells

Cazillis

Giocanti

Houssais

et al. 1984

European Journal of Biochemistry

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“…Although the mechanism of post-termination recycling of ribosomes in eukaryotes is not understood (Janosi et al 1996;Kisselev and Buckingham 2000), eIF3 and, to some extent, eIF1A have been implicated in preventing reassociation of free 40S and 60S subunits and in dissociation of empty 80S ribosomes (Thompson et al 1977;Trachsel and Staehelin 1979;Thomas et al 1980a). Native 40S subunits in the cytoplasm are stably associated with eIF3 (Freienstein and Blobel 1975) and purified eIF3 can bind directly to 40S subunits in the absence of other initiation factors (Benne and Hershey 1976;Peterson et al 1979;Trachsel and Staehelin 1979;Nygard and Westermann 1982). The ability of eIF3 to bind to 40S subunits in the absence of other initiation factors depends strictly on the presence of its loosely associated eIF3j subunit (Fraser et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

Kolupaeva¹,

Unbehaun²,

Lomakin³

et al. 2005

RNA

114

131

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The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j−, respectively). Although unlike eIF3j+, eIF3j− does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2·GTP·Met-tRNA i Met ternary complex, eIF3j−/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j− nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.

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“…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.…”

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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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Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3.small ribosomal subunit, as shown by cross-linking experiments

Cited by 45 publications

References 18 publications

Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells

Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

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

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