The effect of phosphorylation on the functional activity of eukaryotic elongation factor 2 (eEF-2) was studied using a purified phosphorylated factor. The modified factor was unable to stimulate protein synthesis in an eEF-2-dependent rabbit reticulocyte lysate. The functional alteration was further analyzed by measuring the effects of phosphorylation on the ability of the factor to catalyse the ribosome-dependent hydrolysis of GTP. Kinetic analysis showed that both phosphorylated and unmodified factor was able to hydrolyse GTP with approximately the same maximum rate, indicating that the rate of nucleotide exchange was not impaired by the modification. However, the phosphorylated factor showed a marked reduction in the second-order rate constant, suggesting that the phosphorylation interfered with ribosome . eEF-2 complex formation by reducing the affinity of eEF-2 for the ribosome. This assumption was confirmed by direct measurements of the dissociation constants for the ribosomal complexes containing unmodified and phosphorylated eEF-2.

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The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.

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Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.Eukaryotic 18S ribosomal RNA is considerably longer than its prokaryotic homolog 16S rRNA (Clark 1987). Despite differences in size and sequence, 18S rRNA and 16S rRNA share a common structural core (Gutell et al. 1985) in which the additional nucleotides found in 18S rRNA are inserted at specific positions. The inserted nucleotides form extra sequence elements called variable regions (Neefs and De Wachter 1990) or expansion segments (ES; Gerbi 1996). The length and sequence of these expansion segments varies considerably between organisms.18S rRNA contains 12 expansion segments (Gerbi 1996). One of these expansion segments, referred to as ES6, is located in the central domain of 18S rRNA (Fig. 1). With an average length of 250 nucleotides (Neefs and De Wachter 1990), this is the largest expansion segment found in 18S rRNA. ES6 can be divided in two halves based on sequence variability. The 5Ј half exhibits extensive variability, whereas the sequence of the 3Ј half is more conserved. Sequences corresponding to the latter half are absent in eubacterial and archaeal 16S rRNA (Cannone et al. 2002). Several attempts to construct a secondary structure model for the 3Ј half have been made (Nickrent and Sargent 1991;Hancock and Vogler 1998;Wuyts et al. 2000). However, because of the sequence conservation, structure prediction is difficult, and this part of ES6 is therefore left unstructured in the phylogenetic models of 18S rRNA secondary structure ( Fig. 1; Cannone et al. 2002).The location of ES6 within the three-dimensional structure of the yeast 40S subunit has recently been determined using cryo-electron microscopy (Spahn et al. 2001). The expansion segment is located on the back of the so-called body of the 40S subunit (Fig. 1, inset). The segment is seen as two separate densities, one positioned across the back of the 40S body, whereas the second density is located at the side of the lower part of the body with its lower end close to the so-called left foot (Spahn et al. 2001).The left foot contains another expansion segment, called ES3 (Spahn et al. 2001), only found in eukaryotes (Cannone et al. 2002). ES3 is very variable in size and sequence (Gutell et al. 1985;Wuyts et al. 2002), but the phylogenetic data indicate a similar basic structure for ES3 in different organisms ( Fig. 1; Cannone et al...

The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pretranslocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1 -catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 . GTP . aminoacyl-tRNA . ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1-and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.The translational elongation in eukaryotes is mediated through the two elongation factors EF-1 and EF-2 and involves a cyclic series of reactions by which the amino acids are added to the growing polypeptide chain [l, 21. Each cycle can be divided into three basic reaction steps. In the first step cognate aminoacyl-tRNAs are brought to the A-site of the mRNA-programmed ribosome in the form of a ternary complex with EF-1 and GTP [l, 21. During this reaction, GTP is hydrolysed to GDP and inorganic phosphate [3]. This step is followed by the transfer of the nascent peptide in the ribosomal P-site to the newly attached aminoacyl-tRNA [l, 21. Finally, elongation factor EF-2 promotes the translocation of the elongated peptidyl-tRNA from the A-site to the P-site under the hydrolysis of GTP [4]. A common GTPase center on the ribosome, involving the 5s rRNA . L5 complex, has been suggested to participate in both the EF-1-and EF-2-dependent GTP hydrolysis [5].The two elongation factors bind to the ribosome at identical or partially overlapping binding sites [6 -91. Elongation factor EF-1 associates exclusively with post-translocation ribosomes, i. e. ribosomes having their peptidyl-tRNA in the P-site, whereas EF-2 binds only to pre-translocation ribosomes, i.e. ribosomes carrying the peptidyl-tRNA in the Asite [6, 71. We have previously demonstrated that EF-2 forms two types of ribosomal complexes [lo]. In the presence of nonhydrolysable GTP analogues a high-...

Native small ribosomal subunits from rabbit reticulocytes contain all initiation factors necessary for the formation of the mRNA-containing 48S pre-initiation complex. The complex formed in the presence of Met-tRNAf and 125I-labelled globin mRNA was cross-linked with diepoxybutane, and the covalent mRNA-protein complexes were isolated under denaturating conditions. The proteins of the covalent complex were identified as the 110, 95 and 66/64 kDa subunits of eIF-3. In addition, the 24 kDa cap binding protein and the ribosomal proteins S1, S3/3a, S6 and S11 were found covalently linked to the mRNA. Ribosomal proteins S3/3a and S6 were also involved in the ribosomal mRNA-binding domain of reticulocyte polysomes.

Initiation factor eIF-3 from rat liver forms a binary complex with the small ribosomal subunit. Within this complex, 18S ribosomal RNA can be cross--linked to the 66 000 dalton subunit of eIF-3 by treating the complex with a short bifunctional reagent, diepoxybutane, with a distance of 4A between the reactive groups. In binary complexes containing eIF-3 premodified with the heterobifunctional reagent, methyl -p-azido-benzoylaminoacetimidate (10A), the 66 000 dalton subunit of eIF-3 became covalently bound to 18S rRNA after irradiation of the complex with ultraviolet light. The involvement of only one of the eight eIF-3 subunits in the formation of the covalent RNA-protein complexes indicates a highly specific interaction between 18S rRNA and eIF-3 at the attachment site of the factor on the 40S subunit.