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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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 ...