A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants.
Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.
Hagiya
Akiko
A
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
Naganuma
Takao
T
Maki
Yasushi
Y
Ohta
Jun
J
Tohkairin
Yukiko
Y
Shimizu
Tomomi
T
Nomura
Takaomi
T
Hachimori
Akira
A
Uchiumi
Toshio
T
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D043902:Pyrococcus horikoshii / Q000235:genetics / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D019476:Insect Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D012339:RNA, Ribosomal, 28S; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal; C035690:ribosomal phosphoprotein P1; C054675:ribosomal protein P0; D020558:GTP Phosphohydrolases