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.
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 Mg2. 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 Mg2+-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 Mg2+, 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.
Uchiumi
T
T
Department of Biochemistry, Niigata University School of Medicine, Niigata 951 Japan.
Kominami
R
R
D000818:Animals; D001323:Autoantibodies / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015800:Protozoan Proteins; D012313:RNA / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*
D001323:Autoantibodies; C064601:L12E protein, Trypanosoma cruzi; D015800:Protozoan Proteins; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; D012313:RNA; C016700:ribosomal protein L7-L12; D020558:GTP Phosphohydrolases; D008274:Magnesium