Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Abstract

We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg 2 . Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920 -1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg 2؉ -eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg 2؉ , and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.Despite extensive evidence for functional importance of rRNA molecules within the ribosome, it has been difficult to demonstrate biological activities of protein-free rRNA under physiological salt condition. This difficulty appears to be due to involvement of ribosomal proteins that induce and stabilize the higher order structure of rRNA (reviewed by Noller, 1991). Detailed knowledge of rRNA-protein binding and its effect on the RNA conformation is, therefore, important to elucidate the molecular basis of rRNA function. The "GTPase domain" within domain II of Escherichia coli 23 S rRNA is one of the best characterized portions (reviewed by Cundliffe, 1986;Egebjerg et al., 1990;Ryan et al., 1991;Rosendahl and Douthwaite, 1993). This RNA region has been identified as a site of interaction with elongation factor G (Sköld, 1983;Moazed et al., 1988) and with the antibiotic thiostrepton, an inhibitor of elongation factor-dependent processes in protein synthesis (Thompson et al., 1982;Egebjerg et al., 1989). Ribosomal protein L11 and the acidic protein complex L10(L12) 4 cooperatively bind to this RNA domain (Dijk et al., 1979;Beauclerk et al., 1984) and construct a functional site of the 50 S subunit. L11 may participate in induction of a functionally important RNA conformation (Xing and Draper, 1995). On the other hand, the L10(L12) 4 complex appears to affect the RNA conformation through cooperative binding with L11 (Rosendahl and Douthwaite, 1993).The GTPase domain of eukaryotic 28 S rRNA has been also shown to interact with elongation factor 2 (Uchiumi and Kominami, 1994). However, its interaction w...

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