Direct evidence for interaction of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12.
A complex consisting of the acidic phosphoproteins P0, P1, and P2 (P proteins), L12, and RNA fragments was isolated from rat liver ribosomes after treatment with RNase T1 in the presence of EDTA. The complex was reactive with the anti-28 S RNA antibody specific for the highly conserved "GTPase domain" within 28 S rRNA. This suggests an association of these proteins with the RNA domain. To characterize this complex, the P proteins and L12 were isolated and tested for their binding specificity to the RNA by RNase T1 protection and gel retardation assays. Protein L12 and the P protein complex (P complex) both bound to rat 28 S rRNA and protected sequences comprising residues 1859-1921 and 1838-1936, respectively. The sequences overlap each other and lie in the GTPase domain. An in vitro transcript covering residues 1841-1936 of the 28 S rRNA as well as the protected RNA fragments also showed an ability to bind to the P complex and L12, and the binding was cooperative. RNA sequence elements within residues 1841-1936 required for protein binding were defined using site-directed mutagenesis. A unique internal loop including residues 1858 and 1859 and a distinct subregion comprising residues 1867-1914 in this domain were necessary for the binding of the P complex and L12, respectively. These results indicate that P proteins and L12 bind to restricted sites in the GTPase domain and that the complex constitutes the GTPase-related functional site in mammalian ribosomes.
Uchiumi
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T
Department of Biochemistry, Niigata University School of Medicine, Japan.
Kominami
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R
D000818:Animals; D001483:Base Sequence; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D051381:Rats; D006163:Ribonuclease T1 / Q000494:pharmacology; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000648:ultrastructure*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010750:Phosphoproteins; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; D006163:Ribonuclease T1; D020558:GTP Phosphohydrolases