Cross-linking of mRNA to initiation factor eIF-3, 24 kDa cap binding protein and ribosomal proteins SI, S3/3a, S6 and S11 within the 48S pre-initiation complex

Westermann, Peter; Nygård, Odd

doi:10.1093/nar/12.23.8887

Cited by 56 publications

(29 citation statements)

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“…One other explanation for the additional sites could be the differences in salt concentration used in the various footprinting studies. Here we have used a salt concentration optimal for in vitro protein synthesis (3,27) to avoid salt-induced destabilization of the native 40 S subunits during the modification experiments. Thus, the Mg 2ϩ concentration used here is considerably lower than that used during the footprinting of IF-3 on the 16 S rRNA (8,9).…”

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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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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“…Binding to mRNA might therefore be an intrinsic property of yeast eIF3. In support of this, mammalian PRT1/eIF3b and eIF3a/TIF32 can be UV cross-linked to ␤-globin mRNA in 48S PICs formed in vitro (19,45). The N-terminal domain (NTD) of PRT1 contains an RNA recognition motif (RRM), which is an obvious region to mutate in the hope of generating mutants that would implicate eIF3 in scanning by disrupting a putative PRT1-mRNA interaction.…”

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confidence: 93%

Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast

Nielsen

Valášek

Sykes

et al. 2006

Molecular and Cellular Biology

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We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts ؊ phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts ؊ phenotype is exacerbated by tif32-⌬6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1⌬ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

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“…Further information concerning other proteins which interact with mRNA has been obtained from structural studies of preinitiation complexes. Cross-linking of preinitiation complexes containing mRNA indicates a close proximity not only of mRNA and 18 S rRNA [2] but also of mRNA and ribosomal proteins Sl, S3/3a, S6 and Sll [3], of the 5 '-terminal cap structure of mRNA and a 24 kDa protein [1,3] and of mRNA and the 111 kDa, 96 kDa, and 66164 kDa subunits of eIF-3 [3]. The multitude of components of the preinitiation complex in close association with mRNA suggests that the formation of this complex is likely to be a multi-stage process.…”

Section: Discussionmentioning

confidence: 99%

“…In recent studies reovirus mRNA has been cross-linked to a 24 kDa cap binding protein [l] and to 18 S ribosomal RNA [2]. Cross-linking of complexes between native small ribosomal subunits of rabbit reticulocytes and globin mRNA demonstrates a close proximity between mRNA and (i) a 24 kDa cap binding protein, (ii) three subunits (111 kDa, 96 kDa and 66164 kDa) of eukaryotic initiation factor 3 (eIF-3) and (iii) ribosomal proteins Sl, S3/3a, S6, and Sl 1 [3]. Thus, there is evidence for numerous interactions during binding of mRNA to the preinitiation complex.…”

Section: During Initiationmentioning

confidence: 99%

“…It is of interest to compare the stability of the interaction of the 66 kDa subunit with that of the ribosomal proteins S3, S3a, S6 and Sl 1, which are completely split off under the same conditions, although these ribosomal proteins can be also cross-linked to both 18 S rRNA [15] and globin mRNA [3]. This difference may be explained either by a stronger affinity of the 66 kDa subunit to the RNAs or by a higher stability of the protein against denaturating agents.…”

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confidence: 99%

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