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Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan
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Abstract |
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Introduction |
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In the Bombyx mori P protein, binding of P1–P2 dimers to P0 stimulates rRNA binding of P0 (Shimizu et al. 2002). P1 or P2 alone fails to bind efficiently to P0 and to stimulate the rRNA binding of P0 (Shimizu et al. 2002). The interaction between P1 and P2 therefore appears to be a crucial step for efficient assembly of the functional GTPase-associated center at least in animal systems. The eukaryotic pentameric complex P0(P1–P2)2 binds strongly not only to eukaryotic 28 S rRNA but also to prokaryotic 23 S rRNA (Uchiumi et al. 1999). This complex can also replace the prokaryotic counterpart L10(L7/L12)4 complex on the 50 S subunit in vitro and make the prokaryotic subunit accessible to eukaryotic translation factors, but not to prokaryotic factors (Uchiumi et al. 1999, 2002a,). Interestingly, this change in the factor specificity by protein replacement on the ribosome is accompanied by changes in structural features of the 2660 as well as 1070 regions of the 23 S rRNA (Uchiumi et al. 2002b). These previous findings indicate that the P1–P2 interaction also indirectly affects the structure and function of the rRNA domains within the GTPase-associated center.
Although P1 and P2 are different proteins expressed from distinct genes, they share three common structural features. First, the N-terminal half is rich in 
-helical structure (Tchórzewski et al. 2003), which is probably responsible for P1–P2 dimerization and for anchoring them into ribosomal particles through P0 (Jose et al. 1995; Ballesta & Remacha 1996). Second, the middle part is comprised exclusively of alanine, glycine and proline. This region seems to create a very flexible structure. Third, the C-terminal region is rich in hydrophilic amino acids. Of the three regions, the C-terminal regions bear the most striking resemblance to each other. Protein P0 also shares the C-terminal sequence. The sequences of the C-terminal regions are well conserved in eukaryotic organisms and are presumably involved in ribosomal interaction with elongation factors (Uchiumi et al. 1990; Bargis-Surgey et al. 1999).
We report here results of experiments using various truncation mutants of P1 and P2 and also P1/P2 chimeric mutant proteins. These protein variants were used in protein–protein and protein–rRNA binding experiments in vitro to identify amino acid regions within P1 and P2 involved in P1–P2 heterodimerization and ribosomal function. We demonstrate the significance of the N-terminal sequences, not the C-terminal sequences, of both P1 and P2 in P1–P2 heterodimerization and their functional assembly with P0 into the GTPase-associated center.
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Results |
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Silkworm ribosomal proteins P1 and P2 are both comprised of 112 amino acid residues and are structurally homologous to each other (Fig. 1). We constructed plasmids encoding various mutants of P1 and P2 and these are summarized in Fig. 1. P1 mutants included two C-terminal truncation mutants (P1C
26 and P1C52) and four N-terminal truncation mutants (P1N10, P1N20, P1N30 and P1N60). P2 mutants also included two C-terminal truncation mutants (P2C27 and P2C50) and four N-terminal truncation mutants (P2N10, P2N20, P2N30 and P2N62). We also constructed four chimeric proteins (P160–P252, P110–P2102, P262–P150 and P210–P1102) between P1 and P2: P160-P252, comprising amino acid residues 1–60 of P1 fused with residues 61–112 of P2; P110–P2102, residues 1–10 of P1 fused with residues 11–112 of P2; P262–P150, residues 1–62 of P2 fused with residues 63–112 of P1; P210–P1102, residues 1–10 of P2 fused with residues 11–112 of P1. All the protein variants were expressed in E. coli cells and isolated as described in Experimental procedures.
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The formation of P1–P2 heterodimer was examined by a glutathione S transferase (GST)-pulldown assay using GST–P1 or GST–P2 fusion proteins, together with various P1/P2 variants, followed by tricine SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the precipitated proteins (Fig. 2). Effects of truncation of the C-terminal (Fig. 2A) and N-terminal (Fig. 2B) amino acid residues in P1 on the binding to P2 were assayed with GST–P2. The P1 mutants lacking the C-terminal 26 amino acids (P1C26) and 52 amino acids (P1C52) retained the ability to bind to and precipitate GST–P2 (lanes 4 and 6 in Fig. 2A, respectively). In contrast, all P1 mutants lacking the N-terminal 10 amino acids (P1N10), 20 amino acids (P1N20), 30 amino acids (P1N30) and 60 amino acids (P1N60) completely lost the ability to bind to GST–P2 (lanes 4, 6, 8 and 10 in Fig. 2B, respectively). The same experiment was performed to examine the effects of truncation of the C-terminal (Fig. 2C) and the N-terminal (Fig. 2D) parts in P2 on the binding to P1 and the effects assayed with GST–P1. The C-terminal truncation mutants of P2 (P2C27 and P2C50) co-precipitated with GST–P1 (lanes 4 and 6 in Fig. 2C), whereas all the N-terminal truncation mutants were not precipitated (lanes 4, 6, 8 and 10 in Fig. 2D). These results indicate that the N-terminal 10 amino acid residues of both P1 and P2 are important for P1–P2 binding. The same conclusion was derived from other experiments using protein samples without the GST tag sequence where the analysis was performed by native PAGE (data not shown).
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Two P1–P2 heterodimers bind to P0 and form a stable P0.P1–P2 complex (Shimizu et al. 2002; Hagiya et al. 2005). The formation of the P0.P1–P2 complex can be examined by native PAGE (Fig. 3A) where the complex is detected as a clearly shifted band from that of free P1/P2 proteins (lane 1). When the shifted band was cut out of the gel and subjected to SDS-PAGE, followed by immunoblotting with anti-P antibody, all P0, P1 and P2 were detected as the constituents of the complex (data not shown), as previously described (Shimizu et al. 2002). In the gel shown in Fig. 3A, free P0 does not enter the gel. When the C-terminal truncation mutants of P1 (P1C26 and P1C52) were mixed with P2 together with P0, the complex formation was clearly detected (lanes 2 and 3, respectively). Likewise, the complexes were formed when the P2 mutants (P2C27 and P2C50) were mixed with P1 together with P0 (lanes 4 and 5, respectively) and also when two pairs of the C-terminal truncation mutants, P1C26-P2C27 and P1C52-P2C50, were mixed with P0 (lanes 6 and 7, respectively). In contrast, when the N-terminal truncation mutants of P1 (N10, N20, N30 and N60) or P2 (N10, N20, N30 and N62) were mixed with their protein partners together with P0, no shifted band from that of free P1/P2 samples was detected (data not shown), suggesting the truncation of the N-terminal regions of P1 or P2 disrupts the binding ability to P0, which is detectable by the gel analyzes.
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26 (lane 3), P1C52 (lane 4), P2C27 (lane 5), P2C50 (lane 6), P1C26 +P2C27 (lane 7) and even P1C52 +P2C50 (lane 8) retained strong rRNA binding ability that was comparable to that of the intact P0.P1–P2 complex (lane 2). However, neither protein sample containing P1N10 nor P2N10 showed rRNA binding (lanes 9–11). The results indicate that the N-terminal halves of P1 and P2 retain full ability to make P0 bind tightly to the 1070 rRNA domain, whereas the deletion of the N-terminal 10 amino acids in either of P1 or P2 completely disrupts the ability. Activities of hybrid ribosomes containing various truncation mutants of P1 or P2
In order to obtain ribosomal functional data as well as in vitro binding data from the P1 or P2 mutants, we used the hybrid ribosome system, in which eukaryotic P1 and P2 together with P0 replace their E. coli homologous L7/L12 with L10 on the 50 S subunit, as previously described (Uchiumi et al. 2002a). The hybrid ribosomes carrying the intact P1 and P2 proteins (P1WT/P2WT) with P0 showed eEF-2-dependent GTPase activity (Fig. 4A) and eEF-1/eEF-2-dependent polyphenylalanine synthesis (Fig. 4B) at levels comparable to those reported previously (Hagiya et al. 2005). Hybrid ribosomes carrying either C-terminal truncation mutants of P1 (C26 and C52) or P2 (P2C27 and P2C50) showed reduced but significant activity. Even hybrid ribosomes carrying both P1C52 and P2C50, as well as both P1C26 and P2C27, had low levels of activity. In contrast, hybrid ribosomes carrying either P1N10 or P2N10 did not show any activity. These functional data are consistent with the binding data shown in Figs. 2 and 3. The results indicate that the presence of the N-terminal 60 amino acids fragments of both P1 and P2 is sufficient for inducing partial ribosomal activity, while the deletion of the N-terminal 10 amino acids in either P1 or P2 causes the complete loss of ribosome function.
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We also examined the functionally important regions of P1 and P2 without using truncation mutants so as to address the possibility that the truncations might largely alter the tertiary structure of the proteins. We constructed four chimeric P1/P2 proteins (P160–P252, P110–P2102, P262–P150 and P210–P1102, see Fig. 1). Using these chimeric proteins, we performed in vitro binding experiments and functional assays, as described earlier. The chimeric protein P160–P252, comprising P1 sequence in the N-terminal half and P2 sequence in the C-terminal half, bound to GST–P2 (Fig. 5A, lane 2). This chimeric protein efficiently assembled onto the 1070 rRNA domain together with P2 and P0 (Fig. 5B, lane 3) and showed reduced GTPase (Fig. 5C) and polyphenylalanine synthetic (Fig. 5D) activity, comparable to that of the P1C52–P2WT dimer (Fig. 4). The chimeric protein P262–P150, comprising P2 sequence in the N-terminal half and P1 sequence in the C-terminal half, also bound to GST–P1 (Fig. 5A, lane 5). The assembly efficiency of P262–P150 chimeric protein onto the 1070 rRNA (Fig. 5B, lane 4) and its function on the ribosome (Fig. 5C,D) was comparable to that of P1WT–P2C50 (Fig. 4). The chimeric protein P110–P2102 is mostly P2 sequence but the N-terminal 10 amino acids are swapped with those of P1, while P210–P1102 is mostly P1 sequence but the N-terminal 10 amino acids are swapped with those of P2. The P110–P2102 protein bound only weakly to GST–P1 (Fig. 5A, lane 6), and the P210–P1102 protein bound weakly to GST–P2 (lane 3). Moreover, both the chimeric proteins showed no ability to assemble onto the rRNA with P0 (Fig. 5B, lanes 5 and 6) or to enhance the ribosome function (Fig. 5C,D). It should be mentioned that the P110–P2102 and P210–P1102 proteins completely lost the functional dimerization with any other proteins used in this study (Fig. 1B), strengthening the evidence that the heterodimerization of P1 and P2 through interaction between the N-terminal regions is crucial for the functional assembly of the GTPase-associated center.
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Discussion |
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The present results show that the N-terminal ends comprising 10 amino acids in both P1 and P2 are particularly important for the P1–P2 dimerization, whereas the C-terminal halves of both proteins seem not to participate in the dimerization. Our results are consistent with previous data by Gonzalo et al. (2001). They observed that the N-terminal half of rat P1 (residues 1–63) was insoluble but could be solubilized by mixing with the N-terminal half of P2 (residues 1–65). From these results they suggested interaction between these N-terminal halves. Only a limited amount of other data is available about the possible structure of the P1–P2 dimer and the contribution of the N-terminal ends of both proteins to the dimerization. Circular dichroism (CD) spectroscopic data indicate that the yeast P1–P2 dimer contains 65%
-helix (Tchórzewski et al. 2003) and several -helical regions are also predicted within P1 and P2 based on the amino acid sequences (Tchórzewski et al. 2003). The secondary structure prediction indicates that the N-terminal 10 amino acids in both P1 and P2 constitute -helices.
In the case of the eubacterial stalk homodimer L12–L12, the structure of the N-terminal domain encompassing residues 1–30 has been solved using isolated full-length L12 protein by NMR spectroscopy (Bocharov et al. 2004) and as a complex with L10 by X-ray crystallography (Diaconu et al. 2005). Amino acid residues 4–11 and 16–30 of L12 form the first and second -helices and participate in L12–L12 dimerization as parts of a "four-helix bundle" formed between the two proteins. Considering the presence of sets of -helices in the N-terminal regions of P1 and P2 (Zurdo et al. 2000a; Tchórzewski et al. 2003), it is conceivable that eukaryotic P1–P2 dimerization occurs through a four-helix bundle like the eubacterial L12–L12 homodimer, although primary sequence homologues between eukaryotic P1/P2 and eubacterial L12 are very low. In previous work, Tsurugi et al. suggested a binding mechanism between these proteins based on primary sequence alignment and helical wheel analysis (Tsurugi & Mitsui 1991; Tsurugi 1992), that is, a zipper-like structure constructed with hydrophobic amino acids present in P1 and P2. It is possible that the N-terminal helices of P1 and P2 may be directly or indirectly involved in a dimerization motif unique to eukaryotic proteins. Crystal structure data will clarify this point.
The role of the N-terminal regions of P1 and P2 in the assembly of the GTPase-associated center
In a previous in vitro study, we demonstrated the mode of assembly of P0.P1–P2 onto 28 S rRNA, a process important in the formation of the GTPase-associated center (Shimizu et al. 2002; Hagiya et al. 2005). Two P1–P2 heterodimers bind in tandem to the C-terminal region of P0 and then the P0.P1–P2 complex binds to the 1070 rRNA domain. It remains, however, to be clarified which regions of P1 and/or P2 interact with P0 and whether P1 and P2 have independent binding sites for P0. Our present study indicates that the deletion of the N-terminal 10 amino acids of either P1 or P2 disrupts binding of both P1 and P2 to P0. Moreover, the same deletion in P1 or P2 produces proteins that completely fail to stimulate P0 binding to rRNA. In contrast, the deletion of the C-terminal halves of both P1 and P2 causes no disruptive effect on the molecular assembly. The results indicate that the dimer of the N-terminal halves of P1 and P2 is an inseparable unit required for their tight binding to P0 and also for stimulation of P0 binding to rRNA. The assembly mode of P0, P1 and P2 may be analogous to that in eubacterial L10 and L12–L12 dimers, in which the N-terminal domains of L12 molecules tightly associated in a four-helix bundle bind to L10 (Diaconu et al. 2005). Our results, however, are inconsistent with a report by Zurdo et al. (2000b) who used yeast proteins or Gonzalo et al. (2001) who used rat proteins, who have both shown that P1 alone but not P2 alone was able to interact with P0. Our results are also inconsistent with yeast data by Krokowski et al. (2005), who have shown that P1 alone and to some extent P2 alone are able to interact with P0, probably as homodimers. This discrepancy may be due to differences in experimental approaches.
Correlation between P1–P2 dimerization and ribosomal function
As expected from the assembly data, P1 and P2 proteins whose N-terminal 10 amino acids are truncated do not induce ribosomal activity with respect to accessibility of eukaryotic elongation factors at all. The same results are obtained using P1/P2 chimeric proteins P110–P2102 and P210–P1102 (Fig. 1). These results are further evidence that the P1–P2 heterodimerization, through interaction between the N-terminal halves, is crucially important for functional assembly of the GTPase-associated center. This assembly mode is somehow different from that in yeast. Yeast P0 seems to retain the ability to assemble on to the ribosome without P1- and P2-like proteins (Remacha et al. 1995) and induce a low level of ribosomal activity. It has been suggested that the C-terminal sequence common to P0, P1 and P2 has a role in the interaction between ribosomes and elongation factors (Uchiumi et al. 1990; Bargis-Surgey et al. 1999). The result with the yeast ribosome deficient in P1 and P2 suggests that the C-terminal sequence of P0 alone can give a basal level of ribosomal activity (Remacha et al. 1995). The present results suggest that the N-terminal halves of animal P1–P2 are sufficient to make P0 assemble onto ribosomes, but give them only a low level of activity. This level of activity may be similar to that of yeast ribosomes carrying only P0, because, in both cases, ribosomes have a single C-terminal sequence common between P0, P1 and P2. Animal ribosomal P0 probably depends more heavily than yeast P0 on P1–P2 heterodimers for rRNA binding.
In contrast to the drastic effect of the truncation of the N-terminal ends, the truncation of the C-terminal region of either P1 or P2 gives only a minor effect on eEF-2-dependent GTPase (Fig. 4A) and polyphenylalanine synthesis (Fig. 4B) in the presence of P0, including the C-terminal sequence. The truncation of P1 causes a larger effect than that of P2, suggesting that roles of the C-terminal halves in P1 and P2 are not equivalent. The C-terminal half of P1 may be more important for ribosomal function than that of P2. However, the swapping of the C-terminal half of P2 with that of P1 (chimera P262–P150) gives no stimulation effect on ribosomal function (Fig. 5C,D). Heterosequences in P1 and P2 are significant for function not only in the N-terminal regions but also in the C-terminal regions, although the sequences of the C-terminal ends of P1 and P2 are highly similar to each other. Asymmetrical structural features of P1 and P2 seem to be responsible for characteristics of the eukaryotic ribosomal GTPase-associated center.
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Experimental procedures |
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The coding regions for Bombyx mori ribosomal proteins P0, P1, P2 and eL12 (a eukaryotic homologue of prokaryotic L11) were inserted into the E. coli expression vectors pET28c or pET3a (Novagen) and cloned, as previously described (Hagiya et al. 2005). Expression and purification of each ribosomal protein were performed as described (Hagiya et al. 2005). P1 and P2 genes were also cloned into the pGEX-6P-1 vector (GE Healthcare) and expressed as fusion proteins, GST–P1 and GST–P2, in which GST was fused at the N termini of P1 and P2. These fusion proteins were purified according to the manufacture's instructions. The DNA fragments coding for various truncated P1 or P2 proteins (see Fig. 1) were amplified by PCR using cDNAs encoding full-length P1 (WT: 1–112 amino acids) and P2 (WT: 1–112 amino acids) as templates. The DNA fragments coding for the C-terminal truncation mutants of P1 or P2 were inserted into pET28c (Novagen), while the DNA fragments coding for the N-terminal truncation mutants of P1 or P2 were inserted into pGEX-6P-1. The chimeric P1/P2 variants (P160–P252, P110–P2102, P262–P150 and P210–P1102) shown in Fig. 1B were constructed by an overlapping PCR method (Zhong & Bajaj 1993) and inserted into pET28c. The C-terminal truncation mutants of P1 or P2 and P1/P2 chimeric variants were expressed and purified as described above. The GST fusion proteins were expressed using plasmids derived from pGEX-6P-1 and purified on Glutathione Sepharose 4B (GE Healthcare). The GST moiety was removed from the expressed proteins by digestion with PreScission Protease (GE Healthcare). The purity of all proteins was checked by SDS-PAGE.
GST pulldown assay
The GST-fusion proteins were mixed with various non-tagged protein samples at a molar ratio of GST-fusion protein and non-tagged protein of 1 : 3 in the presence of 6 M urea and dialyzed against 0.3 M KCl, 5 mM 2-mercaptoethanol, 20 mM Tris–HCl, pH 7.6 at 4 °C. The dialyzed samples were mixed with a small amount of glutathione Sepharose resin pre-equilibrated with 20 mM Tris–HCl, pH 7.6 and gentle mixing continued at 4 °C for 1 h on a rotary mixer. After mixing, the proteins bound to resins were recovered by centrifugation. The resins were washed five times with 100 µL of 20 mM Tris–HCl, pH 7.6. The resins were then resuspended in SDS sample solution and heated at 95 °C for 5 min. The GST-fusion proteins and the associated protein partners, which were eluted from resins, were separated by tricine SDS-PAGE (Schagger & von Jagow 1987). The gel was stained with Coomassie Brilliant Blue.
P0.P1–P2 complex formation
The P0.P1–P2 complex was reconstituted by mixing of isolated P0, P1 and P2 in the presence of 6 M urea at a molar ratio of 1 : 3 : 3, as previously described (Shimizu et al. 2002). The formation of P0.P1–P2 complex was confirmed by 6% native PAGE (acrylamide/bisacrylamide ratio 39 : 1) at 6.5 V/cm with a buffer system containing 5 mM MgCl2, 50 mM KCl, 50 mM Tris–HCl, pH 8.0. Samples were electrophoresed for 6 h at constant voltage and 4 °C with buffer recirculation (Shimizu et al. 2002). The gel was stained with Coomassie Brilliant Blue.
Gel retardation
The rat rDNA fragment containing residues 1841–1939 of 28S rRNA that correspond to residues 1029–1127 of E. coli 23 S rRNA (designated here as the 1070 domain) was amplified by PCR and inserted into the Hind III and Xba I sites of pT7Blue T-vector (Novagen). The synthesis and purification of the RNA fragment were performed as previously described (Uchiumi et al. 1995) except that T7 RNA polymerase was used instead of SP6 RNA polymerase. An aliquot (10 µL) containing 5 pmol [32P]RNA fragments, 20 mM MgCl2, 0.3 M KCl and 20 mM Tris–HCl, pH 7.6 was preincubated at 65 °C for 5 min and then cooled to 30 °C over 30 min. After addition of P0.P1–P2 complex sample (20 pmol) the mixture was further incubated at 30 °C for 10 min. RNA–protein complexes were separated by 6% native PAGE as described above. The gel was dried and subjected to autoradiography.
Ribosomal subunits, elongation factors and functional assays
Ribosomal 50 S subunits were prepared from the L11-deficient E. coli strain AM68 (Dabbs 1979), as previously described (Uchiumi et al. 2002a). The 50 S core particles deficient both in L10.L7/L12 and L11 were prepared by extraction of the L11-deficient 50 S subunits in a solution containing 50% ethanol and 0.5 M NH4Cl at 0 °C, as previously described (Uchiumi et al. 2002a). Eukaryotic elongation factors eEF-1 and eEF-2 were isolated from pig liver as described by Iwasaki & Kaziro (1979). The P0.P1–P2 complex and its variants carrying mutations in P1 or P2 were mixed with E. coli 50 S cores, together with eL12, to construct the hybrid 50 S particle, as previously described (Uchiumi et al. 2002a). eEF-2-dependent GTPase activity and eEF-1/eEF-2-dependent polyphenylalanine synthesis were assayed according to our previous reports (Uchiumi et al. 2002a; Hagiya et al. 2005).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: uchiumi{at}bio.sc.niigata-u.ac.jp
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References |
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Bargis-Surgey, P., Lavergne, J.P., Gonzalo, P., Vard, C., Filhol-Cochet, O. & Reboud, J.P. (1999) Interaction of elongation factor eEF-2 with ribosomal P proteins. Eur. J. Biochem. 262, 606–611.[Medline]
Bocharov, E.V., Sobol, A.G., Pavlov, K.V., Korzhnev, D.M., Jaravine, V.A., Gudkov, A.T. & Arseniev, A.S. (2004) From structure and dynamics of protein L7/L12 to molecular switching in ribosome. J. Biol. Chem. 279, 17697–17706.
Casiano, C. & Traut, R.R. (1991) Protein topography of Sulfolobus solfataricus ribosomes by cross-linking with 2-iminothiolane. Sso L12e, Sso L10e, and Sso L11e are neighbors. J. Biol. Chem. 266, 21578–21583.
Cundliffe, E. (1986) Involvement of specific portions of ribosomal RNA in defined ribosomal functions: a study utilizing antibiotics. In: Structure, Function, and Genetics of Ribosomes (eds B. Hardesty & G. Kramer), pp. 586–604. New York: Springer-Verlag.
Dabbs, E.R. (1979) Selection for Escherichia coli mutants with proteins missing from the ribosome. J. Bacteriol. 140, 734–737.
Diaconu, M., Kothe, U., Schlunzen, F., Fischer, N., Harms, J.M., Tonevitsky, A.G., Stark, H., Rodnina, M.V. & Wahl, M.C. (2005) Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation. Cell 121, 991–1004.[CrossRef][Medline]
Egebjerg, J., Douthwaite, S.R., Liljas, A. & Garrett, R.A. (1990) Characterization of the binding sites of protein L11 and the L10 (L12)4 pentameric complex in the GTPase domain of 23 S ribosomal RNA from Escherichia coli. J. Mol. Biol. 213, 275–288.[CrossRef][Medline]
Garinos, E., Remacha, M. & Ballesta, J.P.G. (2001) Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk. J. Biol. Chem. 276, 32474–32479.
Gonzalo, P., Lavergne, J.P. & Reboud, J.P. (2001) Pivotal role of the P1 N-terminal domain in the assembly of the mammalian ribosomal stalk and in the proteosynthetic activity. J. Biol. Chem. 276, 19762–19769.
Hagiya, A., Naganuma, T., Maki, Y., Ohta, J., Tohkairin, Y., Shimizu, T., Nomura, T., Hachimori, A. & Uchiumi, T. (2005) 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. J. Biol. Chem. 280, 39193–39199.
Iwasaki, K. & Kaziro, Y. (1979) Polypeptide chain elongation factors from pig liver. Methods Enzymol. 60, 657–676.[Medline]
Jose, M.P., Santana-Roman, H., Remacha, M., Ballesta, J.P. & Zinker, S. (1995) Eukaryotic acidic phosphoproteins interact with the ribosome through their amino-terminal domain. Biochemistry 34, 7941–7948.[CrossRef][Medline]
Kopke, A.K., Leggatt, P.A. & Matheson, A.T. (1992) Structure–function relationships in the ribosomal stalk proteins of archaebacteria. J. Biol. Chem. 267, 1382–1390.
Krokowski, D., Boguszewska, A., Abramczyk, D., Liljas, A., Tchórzewski, M. & Grankowski, N. (2006) Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins. Mol. Microbiol. 60, 386–400.[CrossRef][Medline]
Krokowski, D., Tchórzewski, M., Boguszewska, A. & Grankowski, N. (2005) Acquisition of a stable structure by yeast ribosomal P0 protein requires binding of P1A-P2B complex: in vitro formation of the stalk structure. Biochim. Biophys. Acta 1724, 59–70.[Medline]
Maassen, J., Schop, E.N., Brands, J.H., van Hemert, F.J., Lenstra, J.A. & Möller, W. (1985) Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia. Eur. J. Biochem. 149, 609–616.[Medline]
Nomura, T., Nakano, K., Maki, Y., Naganuma, T., Nakashima, T., Tanaka, I., Kimura, M., Hachimori, A. & Uchiumi, T. (2006) In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits. Biochem. J. 396, 565–571.[CrossRef][Medline]
Remacha, M., Jimenez-Diaz, A., Santos, C., Briones, E., Zambrano, R., Rodriguez-Gabriel, M.A., Guarinos, E. & Ballesta, J.P. (1995) Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects. Biochem. Cell Biol. 73, 959–968.[Medline]
Rich, B.E. & Steitz, J.A. (1987) Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly. Mol. Cell. Biol. 7, 4065–4074.
Schagger, H. & von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.[CrossRef][Medline]
Shimizu, T., Nakagaki, M., Nishi, Y., Kobayashi, Y., Hachimori, A. & Uchiumi, T. (2002) Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA. Nucleic Acids Res. 30, 2620–2627.
Tchórzewski, M., Boldyreff, B., Issinger, O.G. & Grankowski, N. (2000) Analysis of the protein–protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system. Int. J. Biochem. Cell Biol. 32, 737–746.[CrossRef][Medline]
Tchórzewski, M., Krokowski, D., Boguszewska, A., Liljas, A. & Grankowski, N. (2003) Structural characterization of yeast acidic ribosomal P proteins forming the P1A-P2B heterocomplex. Biochemistry 42, 3399–3408.[CrossRef][Medline]
Tsurugi, K. (1992) Hydrophobic zippers and hook-and-eye: evolutionarily conserved protein sequence motifs in eukaryotic acidic ribosomal proteins which are assumed to be involved in the association of the protein family. Protein Seq. Data Anal. 5, 33–38.[Medline]
Tsurugi, K. & Mitsui, K. (1991) Bilateral hydrophobic zipper as a hypothetical structure which binds acidic ribosomal protein family together on ribosomes in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 174, 1318–1323.[CrossRef][Medline]
Uchiumi, T., Honma, S., Endo, Y. & Hachimori, A. (2002a) Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center. J. Biol. Chem. 277, 41401–41409.
Uchiumi, T., Honma, S., Nomura, T., Dabbs, E.R. & Hachimori, A. (2002b) Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts. J. Biol. Chem. 277, 3857–3862.
Uchiumi, T., Hori, K., Nomura, T. & Hachimori, A. (1999) Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding. J. Biol. Chem. 274, 27578–27582.
Uchiumi, T., Traut, R.R. & Kominami, R. (1990) Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes. J. Biol. Chem. 265, 89–95.
Uchiumi, T., Wada, A. & Kominami, R. (1995) A base substitution within the GTPase-associated domain of mammalian 28 S ribosomal RNA causes high thiostrepton accessibility. J. Biol. Chem. 270, 29889–29893.
Wahl, M.C., Bourenkov, G.P., Bartunik, H.D. & Huber, R. (2000) Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12. EMBO J. 19, 174–186.[CrossRef][Medline]
Wool, I.G., Chan, Y.L. & Glück, A. (1995) Structure and evolution of mammalian ribosomal proteins. Biochem. Cell Biol. 73, 933–947.[Medline]
Wool, I.G., Gluck, A. & Endo, Y. (1992) Ribotoxin recognition of ribosomal RNA and a proposal for the mechanism of translocation. Trends Biochem. Sci. 17, 266–269.[CrossRef][Medline]
Zhong, D. & Bajaj, S.P. (1993) A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences. Biotechniques 15, 874–878.[Medline]
Zurdo, J., Gonzalez, C., Sanz, J.M., Rico, M., Remacha, M. & Ballesta, J.P. (2000a) Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity. Biochemistry 39, 8935–8943.[CrossRef][Medline]
Zurdo, J., Parada, P., van den Berg, A., Nusspaumer, G., Jimenez-Diaz, A., Remacha, M. & Ballesta, J.P. (2000b) Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins. Biochemistry 39, 8929–8934.[CrossRef][Medline]
Received: 13 November 2006
Accepted: 10 January 2007
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