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Originally published In Press as doi:10.1074/jbc.M705412200 on September 5, 2007

J. Biol. Chem., Vol. 282, Issue 45, 32827-32833, November 9, 2007
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Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism*Formula

Yasushi Maki{ddagger}1, Tetsuo Hashimoto§, Min Zhou, Takao Naganuma{ddagger}, Jun Ohta{ddagger}, Takaomi Nomura||, Carol V. Robinson, and Toshio Uchiumi{ddagger}2

From the {ddagger}Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan, the §Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki 305-8577, Japan, the Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, and the ||Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan

Received for publication, July 2, 2007 , and in revised form, August 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12)2(Ph-L12)2(Ph-L12)2 was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ribosomal stalk proteins at the "GTPase-associated center" in the large subunits play a central role in the interaction between the ribosomes and GTP-bound translation factors (1-4). In bacteria, the stalk protein is L7/L12 and is usually present in four copies in the form of two stable dimers bound to the C-terminal regions of L10 (5, 6). The L10·L7/L12 complex, together with another protein, L11, binds to the highly conserved domain around 1070 (Escherichia coli numbering is used throughout) of 23 S rRNA through the interaction with the L10 moiety (7, 8). The protein complex constitutes a highly flexible domain in the ribosome (9-15), and the detailed structure within the ribosome has not been resolved by x-ray crystallography (16-21). It has been found that an E. coli ribosome mutant carrying only one L7/L12 dimer retains significant translation activity (6), and more recently, that there are a few bacterial species whose ribosomes have three L7/L12 dimers in a heptameric complex, L10(L7/L12)2(L7/L12)2(L7/L12)2 (22, 23). These findings therefore bring the question as to the significance of multi-stalk dimers in the ribosome into focus.

In eukaryotic ribosomes, two related phosphoproteins P1 and P2 are counterparts of bacterial L7/L12 (24-26). They form heterodimers (27-29), and the two P1-P2 dimers bind to neighboring sites within the C-terminal half of the L10-like stalk base protein P0 (30-32). This pentameric P0·P1-P2 stalk complex can be substituted for E. coli L10·L7/L12 complex in the 50 S subunit and makes the ribosome accessible to eukaryotic elongation factors (33).

In a recent study, we reconstituted the functional stalk complex of the archaeal species Pyrococcus horikoshii from the recombinant constituents archaeal P0 from P. horikoshii (Ph-P0)3 and Ph-L12 (34). It is interesting that the archaeal stalk complex has the ability to make the E. coli ribosome accessible to eukaryotic elongation factors at levels comparable with the eukaryotic stalk complex (34). This result suggests that the functional structures of the stalk complexes are highly conserved between Eukarya and Archaea, although there is a marked difference between them in terms of dimers, i.e. eukaryotic heterodimers and archaeal homodimers. Here, we characterized the P. horikoshii stalk complex and unexpectedly identified the third binding site of the stalk dimer in the expanded region within the archaeal P0. We investigated the functional effects of removing one or two stalk dimers on ribosome function. We also performed sequence alignment and phylogenetic analyses and showed the presence of an expanded homologous sequence in all known species of Archaea, implying that archaeal ribosomes may generally contain three stalk dimers.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of P. horikoshii Ribosomal Proteins—The plasmid construction for P. horikoshii proteins Ph-P0, Ph-L12, and silkworm Bm eL12 (a eukaryotic homologue of bacterial L11) was performed, and their expression and purification were performed as described previously (31, 34). The cDNAs for the C-terminal truncation mutants of the Ph-P0 were derived by PCR. Each DNA fragment was cloned into an E. coli expression vector, pET28 (Novagen), and the resultant plasmid was used for transformation of E. coli strain BL21 to express the proteins. Truncations were confirmed by DNA sequencing. The C-terminal truncation mutants expressed in E. coli cells were purified using the same procedure as used for the WT Ph-P0, as described previously (34). The purified proteins were stored at -80 °C until use.

In Vitro Reconstitution of the Stalk Protein-rRNA Complex—Ph-P0 or truncated variants were mixed with 8-fold higher amounts of Ph-L12, and the complexes were formed as described previously (34). The complex formation was confirmed by 6% polyacrylamide gel electrophoresis (34). The P. horikoshii 23 S rRNA fragment containing residues 1155-1224 (corresponding to residues 1043-1112 of E. coli 23 S rRNA) was synthesized in vitro using template DNA and T7 RNA polymerase (34). Binding of Ph-P0·Ph-L12 complex to the rRNA fragment and its analysis by gel electrophoresis were performed as described previously (34).

Ribosomal 50 S Core and Hybrid 50 S Particles—The E. coli ribosomal 50 S core deficient in L10·L7/L12 and L11 was prepared by extraction of the 50 S subunits from the L11-deficient E. coli mutant AM68 (35) in a solution containing 50% ethanol and 0.5 M NH4Cl at 0 °C, as described previously (36). The P. horikoshii-E. coli hybrid 50 S particle was formed by mixing the E. coli 50 S core with the P. horikoshii ribosomal proteins (34) as described in the legends of Figs. 3 and 5. Formation of the hybrid 50 S particle was confirmed on 3% agarose/0.5% acrylamide composite gels, as described by Hagiya et al. (31).

Quantitative Analysis of Ph-L12 Incorporated into the Ribosome—Isolated Ph-L12 (68 µg) was incubated with 500 units of casein kinase II (New England Biolabs) and [32P]ATP for 120 min at 30 °C in a solution containing 50 mM KCl, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.5. The 32P-labeled Ph-L12 was mixed with non-labeled Ph-L12 (215 µg). The specific radioactivity of the resultant sample was 42 cpm/pmol of protein. This labeled protein was added to the non-labeled Ph-P0 (or truncation mutants), and the complex was reconstituted as described above. For the E. coli 50 S core (30 pmol), excess amounts of the protein sample including 60 pmol of Ph-P0 was added, together with 60 pmol of eL12. The sample was then layered onto 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 at 4 °C for 3 h in a Hitachi P-45 ST rotor. The 50 S fraction was collected, and the amount of the associated 32P-labeled Ph-L12 was estimated by radioactivity.

Mass Spectrometry—The intact complex (1 µg/µl) was buffer-exchanged into 300 mM ammonium acetate (pH 7.6) using micro BioSpin 6 columns (Bio-Rad), and 2-µl aliquots were introduced into the mass spectrometer via gold-coated nanoflow capillaries prepared in-house. Mass spectra were recorded on a QSTAR XL mass spectrometer (MDS Sciex) modified for high mass detection (37), and the conditions within the mass spectrometer were adjusted to preserve non-covalent interactions (38).

Eukaryotic Elongation Factors and the Functional Assays—Eukaryotic eEF-1{alpha} and eEF-2 were isolated from pig liver, as described by Iwasaki and Kaziro (39). The activity of the hybrid ribosome with respect to eEF-2-dependent GTPase activity and eEF-1{alpha}- and eEF-2-dependent polyphenylalanine synthesis was assayed as described previously (34, 40), except that the reaction mixture for the polymerization assay contained 5 pmol of the hybrid 50 S subunit and 25 pmol of the E. coli 30 S subunit, and the reaction was performed 7 min at 37 °C using Phe-specific tRNA.

Phylogenetic Analysis of Eukaryotic and Archaeal P0 and Bacterial L10—The amino acid sequences of P0 from 11 species of Eukarya and 10 species of Archaea as well as the sequences of L10 from 28 species of bacteria were aligned using clustalW followed by manual inspection. The alignment is shown in supplemental Fig. S5. One hundred and three unambiguously aligned sites were selected from the alignment and the data set was subjected to phylogenetic analysis using the program package PHYLIP3.6a. The maximum likelihood method of protein phylogeny in the PROML program was used for searching the best tree, assuming the JTT +{Gamma} model with input sequence order randomized three times and global rearrangements. Maximum likelihood bootstrap analyses (100 replications) were carried out using PROML with the same settings. Bootstrap analyses based on the maximum parsimony method and the neighbor joining method with the maximum likelihood (JTT +{Gamma}) distance were also completed (1000 replications) using the programs PROTPARS and NEIGHBOR. The sequence alignment shown in Fig. 1A is the selected data from Homo sapiens, Saccharomyces cerevisiae, Bombyx mori, and 10 species of Archaea.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In a previous study of the silkworm stalk complex (31), we showed that 1) truncation of the C-terminal 55 amino acid residues of P0 (C{Delta}55) still gives a structure with two P1-P2 stalk dimers; 2) truncation of the C-terminal 65 (C{Delta}65) and 81 (C{Delta}81) residues produces a structure that can only bind one P1-P2 dimer; and 3) truncation of the C-terminal 107 residues (C{Delta}107) does not allow any stalk binding activity (Fig. 1A). From these results, we concluded that the region encompassing residues 210-261 in silkworm P0 participates in the binding of two stalk dimers. The amino acid sequence of this region is conserved in eukaryotic and even in archaeal homologues (Fig. 1A). In Ph-P0, this conserved region corresponds to residues 212-263. It is noticeable that Ph-P0 contains a short inserted sequence, namely residues 272-287 preceding the hydrophilic C-terminal region. This extra sequence is also detected in other archaeal homologues. To investigate whether the 212-263 region of Ph-P0 is responsible for binding of the two stalk Ph-L12 homodimers in a similar manner to the eukaryotic counterparts and whether the inserted short sequence of archaeal P0 participates in the stalk binding, we tested the effects of a series of truncations of the C-terminal half of Ph-P0 (Fig. 1A). Five truncated mutants of Ph-P0 (C{Delta}79, C{Delta}89, C{Delta}105, C{Delta}114, and C{Delta}131), together with Ph-L12, were expressed in E. coli cells, purified (Fig. 1B), and used in the following binding experiments.


Figure 1
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  		FIGURE 1.
Ph-P0 C-terminal truncation series. A, amino acid sequence alignment of the C-terminal region of P0-like proteins from silkworm (Bmo, B. mori), human (Has, H. sapiens), yeast (Sce, S. cerevisiae), and 10 archaeal species (Sac, Sulfolobus acidocaldarius; Pae, Pyrobaculum aerophilum; Ape, Aeropyrum pernix; Mva, Methanococcus vannielii; Mka, Methanopyrus kandleri; Afu, Archaeoglobus fulgidus; Hma, Haloarcula marismortui; Tac, Thermoplasma acidophilum; Pto, Picrophilus torridus; Pho, P. horikoshii). The sites of truncation in the present study are marked below the P0 sequence of P. horikoshii. The sites of truncation in silkworm P0 used in our previous study are indicated above the P0 sequence of silkworm. An extra segment of 16 amino acids, which is observed in Ph-P0 but not in the eukaryotic P0, is underlined. B, SDS gel electrophoretic pattern of isolated protein samples (1 µg each): lane 1, WT Ph-P0; lane 2, C{Delta}79; lane 3, C{Delta}89; lane 4, C{Delta}105; lane 5, C{Delta}114; lane 6, C{Delta}131; lane 7, Ph-L12. The smearing pattern in lane 7 is due to the extremely stable dimerization property of Ph-L12 even in the presence of SDS (34).

 
By adding excess amounts of Ph-L12 to individual Ph-P0 variants, the Ph-P0·Ph-L12 stalk complexes were reconstituted as described under "Experimental Procedures." The formation of the complexes was analyzed by native polyacrylamide gel electrophoresis (Fig. 2A). The complexes were observed as shifted bands of free Ph-L12 (lane 1) by mixing with WT Ph-P0 (lane 2), C{Delta}79 (lane 3), C{Delta}89 (lane 4), C{Delta}105 (lane 5), and C{Delta}114 (lane 6) but not with C{Delta}131 (lane 7). Because 1) all Ph-P0 samples without Ph-L12 were not observed as clear bands (supplemental Fig. S1) and 2) the shifted bands contained Ph-P0, together with Ph-L12, detected by SDS gel electrophoresis (supplemental Fig. S2) and immunoblotting,4 it is highly likely that the Ph-P0·Ph-L12 stalk complexes formed appear as shifted bands. Each reconstituted sample was tested for rRNA binding by a 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 samples. A clear single shifted band in each sample indicates the homogeneity of each complex formed. Moreover, the reconstituted stalk complexes were confirmed to bind to E. coli 50 S core particle deficient in the cognate stalk complex L10·L7/L12 and L11 (supplemental Fig. S3). All these data suggest that binding sites for Ph-L12 stalk dimers lie in the C-terminal 131 amino acids including an inserted 272-287 residues in the Ph-P0 sequence.

To investigate the copy number of Ph-L12 bound to WT Ph-P0 and its truncated variants, Ph-L12 was labeled with 32P by casein kinase II as described under "Experimental Procedures" and used for reconstitution of the stalk complexes. Each Ph-P0 sample (100 pmol each) was mixed with an 8-fold higher amount of 32P-labeled Ph-L12 and reconstituted. The complex formation with each Ph-P0 sample was confirmed by the same native gel as shown in Fig. 2 (see also supplemental Fig. S4). The excess amount of each reconstituted complex was added to a given amount of E. coli 50 S core, which was completely deficient in L10·L7/L12. By sucrose gradient centrifugation, the hybrid particle composed of E. coli 50 S core and the archaeal stalk complex was collected. The labeled Ph-L12 protein included was quantified by measurement of the specific radioactivity of 32P-Ph-L12 (42 cpm/pmol protein), and the value was related to the amounts of 50 S particles estimated by A260 (Fig. 3). In the stalk complex with WT Ph-P0 (Fig. 3A), 5.8 pmol of Ph-L12 were incorporated per 50 S particle (Fig. 3E). In the complex samples with C{Delta}79 (Fig. 3B) and C{Delta}105 (Fig. 3C), 4.1 and 2.0 pmol of Ph-L12 were present in the 50 S particles, respectively (Fig. 3E). No incorporation of Ph-L12 into the 50 S particle with C{Delta}131 was observed (Fig. 3D). Since Ph-L12 tightly forms a homodimer (34), it is highly likely that WT Ph-P0, C{Delta}79, C{Delta}105, and C{Delta}131 bind 3, 2, 1, and 0 Ph-L12 dimers, respectively. The results clearly show that the extra sequence present in Ph-P0 is involved in binding of one of three stalk dimers.


Figure 2
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  		FIGURE 2.
Effect of C-terminal truncation of Ph-P0 on the assembly of Ph-P0 and Ph-L12 onto rRNA. A, the complexes were reconstituted by mixing an excess amount of Ph-L12 with WT-P0 (lane 2), C{Delta}79 (lane 3), C{Delta}89 (lane 4), C{Delta}105 (lane 5), C{Delta}114 (lane 6), and C{Delta}131 (lane 7) as described under "Experimental Procedures." Each sample containing 100 pmol of P0 was subjected to native gel electrophoresis. On the same gel, 800 pmol of Ph-L12 alone was loaded (lane 1). After electrophoresis, the gel was stained with Coomassie Brilliant Blue. B, the same samples as in A containing 20 pmol of Ph-P0 were mixed with 5 pmol of 32P-labeled RNA fragment covering the 1070 domain (lane 1, RNA alone), separated by native gel electrophoresis, and then subjected to autoradiography.

 
The stoichiometry of the WT complex was confirmed by mass spectrometry of the intact protein complex. The charge states series 21+ to 25+ at m/z 4000-5000 enable the mass of the complex to be measured as 106,611 ± 39 Da (Fig. 4). This value is consistent with the heptameric complex with three copies of L12 dimer (calculated mass 106,094 Da).

In a previous study, we observed that the P. horikoshii stalk complex incorporated into E. coli 50 S core functions more efficiently with eukaryotic elongation factors than with the archaeal factors at 37 °C (34). Here we used this hybrid system to investigate the functional role of the three Ph-L12 dimers. The ribosome carrying the C{Delta}105 mutant that bound a single Ph-L12 dimer showed significant GTPase (Fig. 5A) and polyphenylalanine synthetic activities (Fig. 5B). Both the activities corresponded to ~70% of that of the ribosome carrying WT Ph-P0 that could bind three Ph-L12 dimers. The activity of the ribosome with C{Delta}79 that could bind two Ph-L12 dimers was 90-95% of the ribosome carrying WT Ph-P0. The activity of two dimer-ribosome is higher than that of the one-dimer ribosome and slightly lower than the WT Ph-P0-carrying ribosome.


Figure 3
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  		FIGURE 3.
Stoichiometric analysis of Ph-L12 bound to the WT and C-terminal truncation mutants of Ph-P0 incorporated into E. coli 50 S core particles. Ph-L12 was 32P-phosphorylated in vitro by casein kinase II as described previously (31). The labeled Ph-L12 (800 pmol; 42 cpm/pmol of protein) was used in the formation of Ph-P0·Ph-L12 complex, and the samples containing 60 pmol of WT-P0 (A), C{Delta}79 (B), C{Delta}105 (C), and C{Delta}131 (D) were incubated with 30 pmol of 50 S core particles and loaded onto the sucrose density gradient, as described under "Experimental Procedures." The gradients were fractionated after the centrifugation, and the absorbance at 260 nm and the radioactivity of each fraction were monitored. E, the copy numbers of Ph-L12 incorporated into the 50 S cores were derived from three independent experimental data sets including A-D in this figure.

 
To understand how many species have the third binding site for the ribosomal stalk dimer, we compared amino acid sequences of P0-type proteins from 11 Eukarya and 10 Archaea and L10-type proteins from 28 bacteria. The extra sequence observed in Ph-P0 as the third binding site of the stalk dimers exists in all archaeal sequences (Fig. 1A) but not in eukaryotic P0 (supplemental Fig. S5). In bacteria, however, almost a half of known sequences were found to contain the extra sequences in their C-terminal regions. This leads to a suggestion that most of the archaeal ribosomes and many of the bacterial ribosomes contain three stalk dimers, whereas eukaryotic ribosomes contain two.


Figure 4
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  		FIGURE 4.
Electrospray mass spectrum of the wild type P. horikoshii stalk complex. Well resolved charge states (25+ to 21+) are observed, enabling the measured molecular mass to be determined as (106 611 ± 39 Da). This clearly shows that the stalk complex is composed of one copy of P0 and three copies of the L12 dimers. The molecular mass of the heptameric complex is calculated to be 106,094 Da. The small difference in mass between the measured and the calculated value is due to the retention of water molecules and buffer ions under the "soft" ionization conditions employed (43).

 


Figure 5
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  		FIGURE 5.
Relationship between number of Ph-L12 stalk dimers bound to Ph-P0 and ribosome function. A, E. coli 50 S core particles (2.5 pmol) were preincubated without any stalk complex (column 1) or with 20 pmol of the complexes as follows. Column 2, WT-P0·Ph-L12; column 3, C{Delta}79·Ph-L12; column 4, C{Delta}89·Ph-L12; column 5, C{Delta}105·Ph-L12; column 6, C{Delta}114·Ph-L12; column 7, C{Delta}131·Ph-L12. Then particles were assayed for eukaryotic eEF-2-dependent GTPase activity in the presence of silkworm eL12 and E. coli 30 S subunits. B, the same samples (5 pmol of core) as in A were assayed for eukaryotic eEF-1{alpha} and eEF-2 dependent poly(U)-directed polyphenylalanine synthesis. The mean values of the three functional assays were shown in both A and B.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that three stalk dimers bind side by side to the C-terminal half of the anchor protein Ph-P0 at the ribosomal GTPase-associated center in the archaeal species P. horikoshii. The amino acid sequence alignment for the stalk binding sites of P0-like proteins indicates that the region for binding of two stalk dimers are conserved from Archaea to Eukarya (supplemental Fig. S5). The region for the third stalk dimer in the C-terminal region includes an extended sequence, which is present and conserved in all known Archaea but not in Eukarya. The present evidence, together with previous data from the eukaryotic stalk complex (31), implies that archaeal ribosomes may generally contain three stalk dimers and that eukaryotic ribosomes may contain two. This is consistent with a hypothesis based on sequence comparison data by Shimmin et al. (41) that predicts the presence of three modules in archaeal P0-like proteins, which may participate in interaction with stalk dimers. In contrast, the present results are inconsistent with cross-linking data with Sulfolobus solfatarius ribosomes (42) that suggested a pentameric structure containing two stalk dimers. Further extensive studies with various archaeal species will be required to fully understand the situation.

In the case of bacteria, heptameric protein complexes composed of L10 and three dimers of L7/L12 have recently been detected in ribosomes from three thermophiles, namely Thermus thermophilus, Thermatoga maritima, and Thermus aquaticus (22, 23). We have recently confirmed that one of the three dimers is removed from the reconstituted stalk complex by truncation of the short extended sequence observed in the C-terminal region of L10 in T. thermophilus.5 It is therefore highly likely that the extended sequence in L10 from these three bacteria corresponds to the third binding site for the L7/L12 dimer. Since the homologous sequence for this extended segment is observed in about half of bacterial species (supplemental Fig. S5), many species in bacteria may posses ribosomes with three stalk dimers.

Using the alignment data containing 49 sequences (11 Eukarya, 10 Archaea, and 28 bacteria), we performed phylogenetic analysis (Fig. 6). In this figure, the species having the extra sequences in the C-terminal region are shown in bold. All examined P0 proteins from Archaea contain expanded sequences. Such expanded sequence is missing in all the eukaryotic homologues examined, as explained above. All the thermophilic species were found to have the inserted sequences, but it is noticeable that the expanded sequences are also observed in species other than thermophiles, e.g. Rickettsia prowazekii in bacteria and Haloarcula marismortui in Archaea. Bacterial inserted sequences were found in some independent branches, showing that the deletion of the sequences occurred in each branch independently. These considerations of bacterial as well as archaeal stalk dimers lead to a hypothesis that the ribosome in a common ancestor cell might have had three stalk dimers.


Figure 6
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  		FIGURE 6.
The phylogenetic tree of P0/L10-like proteins. The best tree selected by the PROML analysis was shown. Branch lengths are proportional to the estimated number of substitutions. The names and the branches of the species having the extra sequences in the C-terminal region of the homologous proteins are shown in bold. Values for the maximum likelihood (ML) bootstrap analysis are shown on internal branches if the values exceed 50%. For the two internal branches, the common ancestor of Eukarya and the common ancestor of bacteria, bootstrap values for three methods are shown by arrows.

 
There is an important question to be addressed. That is, what is the functional difference between the two and three stalk dimers in the ribosome? Considering the recent cryo-electron microscopy evidence that the C-terminal domain of the stalk directly interacts with prokaryotic elongation factor G (EF-G) (3), higher efficiency of translation elongation may be expected with the three stalk dimers. We tested this point by deletion of the third stalk dimer from the stalk complex derived using the C-terminal truncation mutants of Ph-P0. We assayed the functions by using a hybrid ribosome system, which we recently developed (32, 34). Unexpectedly, the results showed only a slight effect of deletion of the third stalk dimer in the C-terminal region on factor accessibility (Fig. 5). This indicates that the third stalk dimer in Archaea does not play an essential functional role at low temperature. This stalk dimer may play its role at very high temperature such as that around the optimum growing conditions of P. horikoshii (95 °C). It is also conceivable that the three-stalk complex results in additional interactions not only between stalk dimer and Ph-P0 but also between the neighboring stalk dimers on Ph-P0. These cooperative interactions might increase efficiency in assembly of the stalk complex and its stability in extremely unusual conditions.

The present study indicates that factor accessibility of both the archaeal heptameric stalk complex containing three Ph-L12 dimers and its variant pentameric complex containing two Ph-L12 dimers is comparable. Moreover, the accessibility of the archaeal stalk complex to eukaryotic elongation factors is similar to that of the eukaryotic stalk complex containing two P1-P2 heterodimers, at least at 37 °C (34). These data suggest that the pentameric complex form of Ph-P0(Ph-L12)2(Ph-L12)2 and the intact heptameric form of Ph-P0(Ph-L12)2(Ph-L12)2(Ph-L12)2 bear a striking similarity to the eukaryotic P0(P1-P2)(P1-P2) complex in both structure and function. The two stalk dimers seem to work sufficiently well with respect to functional interaction with eukaryotic elongation factors and the coupled events under normal conditions. This may be one of the reasons why eukaryotic ribosomes lost one of the three stalk dimers through deletion of the third stalk binding site on the P0-like protein during evolution.


   FOOTNOTES
 
* This work was supported by Grant-in-Aid for Scientific Research 14035222, a research grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This study was also supported by a grant for the promotion of Niigata University Research Projects. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5. Back

1 Present address: Dept. of Physics, Osaka Medical College, Osaka 569-8686, Japan. Back

2 To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Niigata 950-2181, Japan. Tel.: 81-25-262-7792; Fax: 81-25-262-7792; E-mail: uchiumi{at}bio.sc.niigata-u.ac.jp.

3 The abbreviations used are: Ph-P0, ribosomal protein P0 from P. horikoshii; Ph-L12, ribosomal protein L12 from P. horikoshii; eEF-1{alpha}, eukaryotic elongation factor 1{alpha}; eEF-2, eukaryotic elongation factor 2; eL12, ribosomal protein L12 from B. mori (equivalent to E. coli L11); WT, wild type. Back

4 J. Ohta and T. Uchiumi, manuscript in preparation. Back

5 T. Nomura, M. Nakatsuchi, and T. Uchiumi, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Kohji Nakano for the help in protein preparation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Möller, W., and Maassen, J. A. (1986) in Structure, Function, and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 309-325, Springer-Verlag New York Inc., New York
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