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.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.

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The involvement of ribosomal RNA in the binding of eukaryotic elongation factor eEF-2 to the ribosome was investigated. eEF-2 was complexed to empty reassociated 80S ribosomes in the presence of the nonhydrolyzable GTP analogue GuoPP[CH2]P. The formed complex was treated with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and micrococcus nuclease to allow specific modification at single-stranded regions of the rRNAs. The sites of modification were localized by primer extension using complementary deoxynucleotide primers and reverse transcriptase. The modification pattern was compared to that obtained from 80S ribosomes lacking bound eEF-2. Binding of the factor to the ribosome resulted in the protection of specific sites in both 18S and 28S rRNA, while the reactivity of 5.8S rRNA was unchanged. In 18S rRNA, the affected nucleotides were localized to the 5'- and 3'-domains, and in 28S rRNA the protected nucleotides were seen in domains II, IV, and V. The alpha-sarcin/ricin loop in domain VI of 28S rRNA was inaccessible for chemical modification even in the absence of bound eEF-2. However, the bound factor protected A4256, located in the alpha-sarcin/ricin loop, from ricin-induced depurination.

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