A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m506050200
ISSN1083-351X
AutoresAkiko Hagiya, Takao Naganuma, Yasushi Maki, Jun Ohta, Yukiko Tohkairin, Tomomi Shimizu, Takaomi Nomura, Akira Hachimori, Toshio Uchiumi,
Tópico(s)Viral Infections and Immunology Research
ResumoRibosomal 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 CΔ65 and CΔ81 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 CΔ107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NΔ21-NΔ92 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 NΔ6, NΔ14, and CΔ18-CΔ81, 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 32P-labeled P1/P2 into the 50 S subunits with WT-P0 and CΔ81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CΔ81 bound only one copy each. The hybrid ribosome with CΔ81 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. 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 CΔ65 and CΔ81 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 CΔ107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NΔ21-NΔ92 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 NΔ6, NΔ14, and CΔ18-CΔ81, 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 32P-labeled P1/P2 into the 50 S subunits with WT-P0 and CΔ81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CΔ81 bound only one copy each. The hybrid ribosome with CΔ81 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. The ribosomal large subunits from all organisms contain an active site termed the "GTPase-associated center" that is responsible for the GTPase-related events in protein biosynthesis. This active site is composed of the two highly conserved domains around 1070 and 2660 (Escherichia coli numbering is used throughout) of 23 S/28 S rRNA and the ribosomal proteins bound to the 1070 region (1Cundliffe E. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 586-604Google Scholar, 2Wool I.G. Gluck A. Endo Y. Trends Biochem. Sci. 1992; 17: 266-269Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 3Egebjerg J. Douthwaite S.R. Liljas A. Garrett R.A. J. Mol. Biol. 1990; 213: 275-288Crossref PubMed Scopus (123) Google Scholar). The protein components of this site in prokaryotic ribosomes constitute a characteristic pentameric complex, L10(L7/L12)2(L7/L12)2 (4Pettersson I. Hardy S.J. Liljas A. FEBS Lett. 1976; 64: 135-138Crossref PubMed Scopus (115) Google Scholar, 5Gudkov A.T. Tumanova L.G. Venyaminov S.Y. Khechinashvilli N.N. FEBS Lett. 1978; 93: 215-218Crossref PubMed Scopus (41) Google Scholar), in which two L7/L12 homodimers bind to the C-terminal regions of L10 (6Griaznova O Traut R.R. Biochemistry. 2000; 39: 4075-4081Crossref PubMed Scopus (33) Google Scholar) and constitute a highly flexible and functionally important lateral protuberance, the so-called "stalk" (7Möller W. Maassen J.A. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 309-325Google Scholar). Although the ribosomal stalk is observed by cryo-electron microscopy (8Agrawal R.K. Linde J. Sengupta J. Nierhaus K.H. Frank J. J. Mol. Biol. 2001; 311: 777-787Crossref PubMed Scopus (101) Google Scholar), the detailed structure of this pentameric complex has not been resolved by x-ray crystallography of ribosomes (9Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2813) Google Scholar, 10Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T Cate J.H. Noller H.F. Science. 2001; 292: 883-896Crossref PubMed Scopus (1672) Google Scholar, 11Harms J. Schluenzen F. Zarivach R. Bashan A. Gat S. Agmon I. Bartels H. Franceschi F. Yonath A. Cell. 2001; 107: 679-688Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). The chemical features of protein-protein and protein-rRNA interactions in the GTPase-associated center remain to be clarified. The animal ribosomal phosphoproteins P0 and P1/P2 (P proteins) are counterparts of prokaryotic L10 and L7/L12, respectively, although P1 and P2 are related but different proteins, unlike prokaryotic L7/L12 (12Maassen J. Schop E.N. Brands J.H. van Hemert F.J. Lenstra J.A. Möller W. Eur. J. Biochem. 1985; 149: 609-616Crossref PubMed Scopus (41) Google Scholar, 13Rich B.E. Steitz J.A. Mol. Cell. Biol. 1987; 7: 4065-4074Crossref PubMed Scopus (235) Google Scholar, 14Wool I.G. Chan Y.-L. Glück A. Biochem. Cell Biol. 1995; 73: 933-947Crossref PubMed Scopus (289) Google Scholar). In yeast cells, there are two P1-type proteins, P1α and P1β, and two P2-type proteins, P2α and P2β (15Ballesta J.P.G. Guarinos E. Zurdo J. Parada P. Nusspaumer G. Lalioti V.S. Perez-Fernandez J. Remacha M. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. ASM Press, Washington, D. C.2000: 115-125Google Scholar). It is believed that P proteins constitute a pentameric complex, designated here as P0·P1-P2, in the GTPase-associated center of eukaryotic ribosomes (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar, 17Hanson C.L. Videler H. Santos C. Ballesta J.P.C. Robinson C.V. J. Biol. Chem. 2004; 279: 42750-42757Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This complex binds not only to eukaryotic 28 S rRNA but also cross-binds to prokaryotic 23 S rRNA (18Uchiumi T. Hori K. Nomura T. Hachimori A. J. Biol. Chem. 1999; 274: 27578-27582Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and determines the specificity of the ribosome for eukaryotic elongation factor 1α (eEF-1α) 2The abbreviations used are:eEF-1αeukaryotic elongation factor 1αeEF-2eukaryotic elongation factor 2SDSsodium dodecyl sulfateeL12eukaryotic ribosomal protein L12 (equivalent to E. coli L11)WTwild typeBmP0B. mori P0 and 2 (eEF-2) (19Uchiumi T. Honma S. Nomura T. Dabbs E.R. Hachimori A. J. Biol. Chem. 2002; 277: 3857-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This strong dependence on the P0·P1-P2 complex for the factor accessibility suggests the direct interaction between the protein complex and elongation factors. It has also been suggested that the P0·P1-P2 complex modulates the functional structures of the sarcin/ricin domain around 2660 as well as the 1070 regions of 23 S/28 S rRNA and makes them accessible to eukaryotic elongation factors (20Uchiumi T. Honma S. Endo Y. Hachimori A. J. Biol. Chem. 2002; 277: 41401-41409Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Knowledge of the molecular details on the assembly of P0, P1, and P2 proteins onto rRNA is essential to clarify protein-dependent function of the GTPase-associated center. eukaryotic elongation factor 1α eukaryotic elongation factor 2 sodium dodecyl sulfate eukaryotic ribosomal protein L12 (equivalent to E. coli L11) wild type B. mori P0 Current biochemical and genetic evidence indicates that P1 and P2 proteins form the heterodimer (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar, 21Tchórzewski M. Boldyreff B. Issinger O.G. Grankowski N. Int. J. Biochem. Cell Biol. 2000; 32: 737-746Crossref PubMed Scopus (53) Google Scholar, 22Garinos E. Remacha M. Ballesta J.P.G. J. Biol. Chem. 2001; 276: 32474-32479Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Gonzalo P. Lavergne J.P. Reboud J.P. J. Biol. Chem. 2001; 276: 19762-19769Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Tchórzewski M. Krokowski D. Boguszewska A. Liljas A. Grankowski N. Biochemistry. 2003; 42: 3399-3408Crossref PubMed Scopus (34) Google Scholar) and P1-P2 dimers bind to a specific region within the C-terminal domain of P0 (25Santos C. Ballesta J.P. J. Biol. Chem. 1995; 270: 20608-20614Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 26Lalioti V.S. Perez-Fernandez J. Remacha M. Ballesta J.P. Mol. Microbiol. 2002; 46: 719-729Crossref PubMed Scopus (33) Google Scholar, 27Perez-Fernandez J. Remacha M. Ballesta J.P. Biochemistry. 2005; 44: 5532-5540Crossref PubMed Scopus (24) Google Scholar). On the other hand, the rRNA binding site seems to be located within the N-terminal region comprising about 200 amino acids (25Santos C. Ballesta J.P. J. Biol. Chem. 1995; 270: 20608-20614Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), although direct evidence has not been shown. In the case of mammalian counterparts, isolated P0 is insoluble in aqueous solution, but the P1-P2 binding to P0 makes P0 soluble (23Gonzalo P. Lavergne J.P. Reboud J.P. J. Biol. Chem. 2001; 276: 19762-19769Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 28Uchiumi T. Kominami R. J. Biol. Chem. 1992; 267: 19179-19185Abstract Full Text PDF PubMed Google Scholar). We recently showed that silkworm P0, however, is soluble and useful for biochemical assays (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). By using the silkworm proteins, we demonstrated that binding of P1 and P2 to P0 induced the binding activity of P0 to rRNA (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). It is therefore conceivable that binding of P1-P2 to P0 at its C-terminal region affects the overall structure of P0. To clarify the individual binding sites for two P1-P2 dimers and for rRNA on P0 in vitro, we here constructed 11 kinds of truncation mutants of silkworm P0 and used them for protein-protein and protein-rRNA binding experiments. We identified two neighboring sites for P1-P2 heterodimers within the C-terminal half and a crucially important site for rRNA binding at the N terminus of P0 and suggested that the protein-protein and protein-RNA bindings mutually affect each other. To evaluate the in vitro binding data on the basis of ribosome function, we used a hybrid ribosome system developed previously (19Uchiumi T. Honma S. Nomura T. Dabbs E.R. Hachimori A. J. Biol. Chem. 2002; 277: 3857-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) in which E. coli L10·L7/L12 complex and L11 on the 50 S subunit were replaced with the eukaryotic counterparts P0·P1-P2 complex and eL12, respectively. Whenever efficient RNA binding could be observed, complexes containing the truncated P0 mutants bound to E. coli core ribosomes and induced activities dependent on eukaryotic elongation factors. It is interesting that ribosomes carrying a P0 variant accessible to only one P1-P2 heterodimer retained reduced but significant activity. Plasmid Construction, Protein Expression, and Purification—The cDNAs for Bombyx mori ribosomal proteins P0, P1, P2, and eL12 (a eukaryotic homologue of prokaryotic L11) were provided by Dr. K. Mita (National Institute of Agrobiological Sciences). The coding region in each cDNA was amplified by PCR (29Saiki R.K. Gelfand D.H. Stoffel S. Sharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13517) Google Scholar), inserted to E. coli expression vector pET28c or pET3a (Novagen), and cloned. Proteins expressed in E. coli cells were purified, as described previously (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The DNA fragments coding for various truncated P0 were also amplified by PCR using cDNA encoding full-length (WT, 1-316 amino acids) of P0 as a template (see Fig. 1). The overlapping PCR method (30Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar) was used to construct the chimeric P0 mutants composed of the N-terminal 1-235 (CΔ81) and 1-209 (CΔ107) amino acid sequences of silkworm P0 fused to the C-terminal sequences of the archaebacterial (Pyrococcus horikoshii) P0-like protein, which are homologous to the 236-316 and 210-316 sequences of silkworm P0, respectively. The genomic DNA was used as a template of PCR to amplify the DNA fragments for the two C-terminal amino acid sequences of P. horikoshii P0. 3Y. Maki, J. Ohta, and T. Uchiumi, manuscript in preparation. Each DNA fragment was cloned into pET28c, and the P0 mutant was expressed and purified as described above. In Vitro RNA Synthesis—The rat rDNA fragment containing residues 1841-1939 that correspond to 1029-1127 of E. coli 23 S (designated here the 1070 domain) was amplified by PCR and inserted into the HindIII and XbaI sites of pSPT 18 (Roche Applied Science). The RNA fragment was synthesized using the plasmid DNA and SP-6 RNA polymerase and purified, as described previously (31Uchiumi T. Wada A. Kominami R. J. Biol. Chem. 1995; 270: 29889-29893Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). P0·P1-P2 Complex Formation—After the amounts of isolated proteins were determined with the Micro BCA protein assay reagent kit (Pierce), the concentrations of individual protein samples (pmol/μl) were estimated considering that 1 μg of WT-P0 (or P0 mutants), P1, and P2 correspond to 29.3 (or 26.6-42.2), 87.3, and 86.6 pmol, respectively. The protein samples were mixed together at a molar ratio of P0 sample: P1:P2 of 1:3:3, and the complex was reconstituted, as described previously (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The P0·P1-P2 complex formation in the presence or absence of a 2-fold excess of the rRNA fragments of the 1070 domain was confirmed by 6% polyacrylamide (acrylamide/bisacrylamide ratio 39:1) native gel electrophoresis at 6.5 V/cm with a buffer system containing 5 mm MgCl2, 50 mm KCl, and 50 mm Tris-HCl (pH 8.0). Samples were electrophoresed for 6 h at constant voltage and 4 °C with buffer recirculation (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The gel was stained with Coomassie Brilliant Blue. Ribosomal Subunits and the 50 S Core Particles—E. coli ribosomal subunits were prepared as described previously (20Uchiumi T. Honma S. Endo Y. Hachimori A. J. Biol. Chem. 2002; 277: 41401-41409Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). 50 S cores deficient in L10·L7/L12 and L11 were prepared by extraction of the 50 S subunits from the L11-deficient E. coli mutant AM68 (32Dabbs E.R. J. Bacteriol. 1979; 140: 734-737Crossref PubMed Google Scholar) in a solution containing 50% ethanol and 0.5 m NH4Cl solution at 0 °C, as described previously (19Uchiumi T. Honma S. Nomura T. Dabbs E.R. Hachimori A. J. Biol. Chem. 2002; 277: 3857-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Gel Retardation—[32P]RNA fragments covering the 1070 domain (5 pmol) synthesized as described above were mixed with 10 pmol of P0·P1-P2 complex sample and 10 pmol of eL12 (28Uchiumi T. Kominami R. J. Biol. Chem. 1992; 267: 19179-19185Abstract Full Text PDF PubMed Google Scholar) and incubated at 30 °C for 5 min in 10 μl of a solution containing 20 mm MgCl2, 300 mm KCl, 20 mm Tris-HCl, pH 7.6 (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The RNA-protein complexes were separated by 6% polyacrylamide native gel, as described above. The gel was dried and subjected to autoradiography. Acrylamide/Agarose Composite Gel Electrophoresis—50 S core particles (10 pmol) were mixed with various P0·P1-P2 complex samples (20 pmol each) together with eL12 (20 pmol). The samples were analyzed by electrophoresis on acrylamide/agarose composite gel composed of 3% acrylamide (acrylamide/bisacrylamide ratio 19:1) and 0.5% agarose (33Tokimatsu H. Strycharz W.A. Dahlberg A.E. J. Mol. Biol. 1981; 152: 397-412Crossref PubMed Scopus (45) Google Scholar), as described previously (19Uchiumi T. Honma S. Nomura T. Dabbs E.R. Hachimori A. J. Biol. Chem. 2002; 277: 3857-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The gel was stained with Azur B. Binding of proteins to the 50 S core particles was revealed by gel mobility shift. Quantitative Analysis of P1 and P2 Incorporated into the Ribosome— Isolated P1 and P2 (25 μg of each) were incubated with 500 units of casein kinase II (New England Biolabs) and 5 nmol of [32P]ATP (250 μCi/μmol) for 30 min at 30 °C in a solution (50 μl) containing 50 mm KCl, 10 mm MgCl2, 20 mm Tris-HCl, pH 7.5. The 32P-labeled P1 (86 cpm/pmol) was mixed with non-labeled P0 (or CΔ81) and P2, and the complex was reconstituted as described above. For the E. coli 50 S core (78 pmol), excess amounts (195 pmol) of the P0·[32P]P1-P2 complex were added, together with 154 pmol of eL12. The sample was then layered on a 10-28% sucrose gradient in a solution containing 50 mm NH4Cl, 5 mm MgCl2, 5 mm 2-mercaptoethanol, and 20 mm Tris-HCl, pH 7.6, and fractionated after centrifugation at 40,000 rpm and 4 °C for 3 h in a Hitachi P-45 ST rotor. The 50 S fraction was collected, and the amount of the associated P1 was estimated by its radioactivity. The 32P-labeled P2 (105 cpm/pmol) was mixed with non-labeled P0 (or CΔ81) and P1, and its incorporation into 50 S core particles was analyzed as described for 32P-labeled P1. Ribosome Functional Assays—Eukaryotic elongation factors eEF-1α and eEF-2 were isolated from pig liver as described by Iwasaki and Kaziro (34Iwasaki K. Kaziro Y. Methods Enzymol. 1979; 60: 657-676Crossref PubMed Scopus (45) Google Scholar). eEF-2-dependent GTPase and polyphenylalanine synthesis by using poly(U), eEF-1α, and eEF-2 were assayed according to our previous report (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). Preparation of Various Truncated Mutants of Animal Ribosomal Protein P0—The eukaryotic ribosomal protein P0 plays a central role in the assembly of the active GTPase-associated center. Here we investigated the structural elements required for binding of two P1/P2 dimers and rRNA by using truncated mutants that are summarized in Fig. 1A. All P0 mutants including five C-terminal mutants (CΔ18, CΔ55, CΔ65, CΔ81, and CΔ107) and six N-terminal truncation mutants (NΔ6, NΔ14, NΔ21, NΔ48, NΔ66, and NΔ92) were expressed in E. coli cells but found to be insoluble. The proteins were solubilized in 6 m urea and purified by using ion exchange high pressure liquid chromatography. The purity of all the isolated P0 samples, P1, P2, and eL12, is shown in Fig. 1B. By adding P1 and P2 to individual P0 samples, P0·P1-P2 complexes were reconstituted as described under "Materials and Methods" and used in the following experiments. Effect of the C-terminal Truncation of P0 on P0·P1-P2 Assembly—The formation of P0·P1-P2 complexes was examined by native polyacrylamide gel electrophoresis (Fig. 2A). The complex of WT-P0 with P1 and P2 was detected as a shifted band (Fig. 2A, lane 1), in a similar manner as the authentic proteins from silkworm ribosomes (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The complexes were also formed efficiently even with P0 mutants, the C-terminal amino acids of which, 299-316 (CΔ18, lane 2) and 262-316 (CΔ55, lane 3), were truncated. However, only weak smearing bands were observed in the complexes with the CΔ65 (lane 4) and CΔ81 (lane 5) mutants of P0. No complex formation was detected in the sample with the CΔ107 mutant (lane 6). Each protein mixture was tested for rRNA binding by gel mobility shift assay using a small amount of the 32P-labeled RNA fragment covering the 1070 region (Fig. 2B). Strong RNA binding was observed for all reconstituted complexes (Fig. 2B, lanes 2-6). The CΔ107 mutant, which had no binding ability to P1/P2, showed reduced but significant binding affinity to the RNA (lane 7). The same mobility shift was observed in the isolated CΔ107 without P1 and P2 (not shown), suggesting that the CΔ107 mutant retains rRNA binding. This is an unexpected result because the WT-P0 fails to bind rRNA without P1 and P2 (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar). The same experiment as Fig. 2A was performed after the addition of an excess amount of the rRNA fragment (Fig. 2C). In the presence of the rRNA fragment, bands of the P0·P1-P2 complexes with all the P0 samples except CΔ107 were more distinct (Fig. 2C) than those in the absence of the RNA (Fig. 2A). Particularly, the complexes with CΔ65 and CΔ81 were stabilized markedly with the RNA fragment (Fig. 2C, lanes 4 and 5). The complex formation of CΔ107 with P1-P2 was not detected even after the addition of the RNA (lane 6). To confirm that the complexes formed in Fig. 2C all contain P0 (or its mutants), P1 and P2, the bands of the complexes were cut out of the gel and subjected to SDS gel electrophoresis. The gel was stained with a fluorescent dye, SYPRO Orange, to quantitate roughly the relative amounts of P1/P2 by fluorescence intensity (Fig. 2D). The three protein components were detected in all complexes formed. The intensity of both P1 and P2 in the complexes with CΔ65 and CΔ81 (lanes 4 and 5) was approximately half of that in the other complexes (lanes 1-3). To eliminate a possibility that the effects of the truncation shown in Fig. 2 result from severe alteration of the tertiary structure of P0 rather than from deletion of the binding sites for P1/P2, we also performed binding experiments using chimeric P0 instead of truncation mutants. Because archaebacterial (P. horikoshii) ribosomes contain a eukaryotic P0-like protein that does not cross-bind to silkworm P1/P2, we replaced the C-terminal 81- and 107-amino acid sequences of silkworm P0 with the homologous sequences of P. horikoshii P0. As shown in Fig. 3, chimeric P0 samples did not form stable complexes with silkworm P1/P2 in the absence of the rRNA fragments (lanes 2 and 3). In the presence of the rRNA fragment, however, the chimeric P0, the C-terminal 81-amino acid sequence of which is from the corresponding region of P. horikoshii protein, could form a complex with silkworm P1/P2 (lane 5). In contrast, the chimeric P0, the C-terminal 107-amino acid sequence of which is from P. horikoshii, had no ability to bind silkworm P1/P2 (lane 6). The abilities of chimeric P0 mutants to complex with P1/P2 are comparable with those of the CΔ81 and CΔ107 P0 mutants (Fig. 2C). It should be added that the chimeric P0, the C-terminal 107-amino acid sequence of which is from P. horikoshii, could bind the P. horikoshii stalk dimers and form a functional complex.3 These results supported the binding data with the C-terminal truncation mutants. Effect of the N-terminal Deletion of P0 on P0·P1-P2 Assembly—The formation of P0·P1-P2 complexes was also examined with the N-terminal truncation mutants of P0 (Fig. 4A). Unlike the C-terminal truncation, all the N-terminal mutants NΔ6-NΔ92 formed complexes that appeared as distinct bands in the presence of P1-P2 (lanes 2-7). The SDS gel electrophoretic analysis of the complexes showed that all contained P0 (or its mutants), P1, and P2 (data not shown), suggesting that the N-terminal truncations did not disrupt the binding potentials between P0 and P1-P2. In contrast, the N-terminal deletions caused marked effect of the rRNA binding (Fig. 4B). When N-terminal amino acids 1-6 (NΔ6) were truncated, the rRNA binding ability of P0·P1-P2 complex was reduced (Fig. 4B, lane 3). By further deletions (NΔ14-NΔ92, lanes 4-8), the binding ability was lost. However, the rRNA bindings of the complexes with NΔ6 and NΔ14, but not with NΔ21-NΔ92, were recovered by the addition of eL12 (data not shown). Assembly of the Truncated P0 Mutants onto the E. coli 50 S Particles— Unlike prokaryotic L10, eukaryotic P0 was hardly released from the animal 60 S subunit by standard high salt/ethanol conditions, and reconstitution experiments of the GTPase-associated center with animal ribosomes are much harder than those with E. coli ribosomes. We have established conditions to form a hybrid ribosomal particle in which L10·L7/L12 complex and L11 within the E. coli 50 S subunit are replaced with animal P0·P1-P2 and eL12, respectively (19Uchiumi T. Honma S. Nomura T. Dabbs E.R. Hachimori A. J. Biol. Chem. 2002; 277: 3857-3862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We therefore used the E. coli 50 S subunits to investigate the assembly of animal P0·P1-P2 and eL12 forming an active GTPase-associated center. The formation of hybrid 50 S particles by mixing E. coli 50 S cores with various truncated P0 mutants, P1/P2, and eL12 was confirmed by acrylamide/agarose composite gel electrophoresis (Fig. 5). The gel mobility of E. coli 50 S cores (Fig. 5, A and B, lanes 1) was shifted upwards by the addition of only recombinant silkworm eL12 (Fig. 5, A and B, lanes 2) and then supershifted by the further addition of WT-P0·P1-P2 complex (Fig. 5, A and B, lanes 3), indicating efficient binding of both proteins to E. coli 50 S core particles. Likewise, the complexes with the C-terminal truncation mutants of P0 (CΔ18-CΔ81) strongly bound to the core particles (Fig. 5A, lanes 4-7), whereas a clear supershift was not detected by the addition of CΔ107 (lane 8). In the case of the complexes with the N-terminal deletion mutants of P0, the supershifts were observed only in the NΔ6- and NΔ14-containing complexes (Fig. 5B, lanes 4 and 5). By further deletions of P0, the ability of ribosome binding was completely lost (lanes 6-9). As shown in Fig. 2A, there is a notable difference in P1/P2 binding property between a group including WT-P0, CΔ18, and CΔ55 and another group of CΔ65 and CΔ81. The former members could bind efficiently P1/P2 without the RNA fragment, whereas the latter mutants required the rRNA fragment. To quantitate the amounts of P1 and P2 bound to P0 and compare them between the two groups, P1 and P2 were labeled in vitro by 32P phosphorylation and then incorporated into the hybrid 50 S particles with WT-P0 (Fig. 6, A and B, left) and CΔ81 (Fig. 6, A and B, right). After sucrose gradient centrifugation, the hybrid particles were collected, and the incorporated P1 and P2 were quantified. When WT-P0 was used, 2.2 copies of P1 (Fig. 6A, left) and 1.7 copies of P2 (Fig. 6B, left) were incorporated per 50 S particle. However, when CΔ81 was used, only 1.1 copies of P1 (Fig. 6A, right) and 1.0 copy of P2 (Fig. 6B, right) were present on the 50 S particles. The results are consistent with those in Fig. 2D and indicate that the WT-P0 binds two copies of both P1 and P2 but that CΔ81 binds only one copy each. Considering previous evidence that P1 and P2 form a heterodimer (16Shimizu T. Nakagaki M. Nishi Y. Kobayashi Y. Hachimori A. Uchiumi T. Nucleic Acids Res. 2002; 30: 2620-2627Crossref PubMed Scopus (67) Google Scholar, 21Tchórzewski M. Boldyreff B. Issinger O.G. Grankowski N. Int. J. Biochem. Cell Biol. 2000; 32: 737-746Crossref PubMed Scopus (53) Google Scholar, 22Garinos E. Remacha M. Ballesta J.P.G. J. Biol. Chem. 2001; 276: 32474-32479Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Gonzalo P. Lavergne J.P. Reboud J.P. J. Biol. Chem. 2001; 276: 19762-19769Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Tchórzewski M. Krokowski D. Boguszewska A. Liljas A. Grankowski N. Biochemistry. 2003; 42: 3399-3408Crossref PubMed Scopus (34) Google Scholar) together with the present data, we infer that WT-P0, CΔ18, and CΔ55 bind two heterodimers, but CΔ65 and CΔ81 bind onl
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