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1 Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake, Sakyo, Kyoto 606-8502, Japan
2 The Institute for Virus Research, Kyoto University, Shogoin-Kawaracho, Sakyo, Kyoto 606-8507, Japan
3 Department of Physics, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
4 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Konoe, Yoshida, Sakyo, Kyoto, 606-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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It was reported that the B. subtilis Obg protein binds to ribosomal protein L13 (Scott et al. 2000) and is required for stress-dependent activation of transcription factor 
B (Scott & Haldenwang 1999). The Obg protein in C. crescentus, named CgtAc, was also found to be associated with the large ribosomal subunit (Lin et al. 2004). The yeast Nog1 protein whose N-terminal half is homologous to the Obg protein was shown to be required for the biogenesis of the large ribosomal subunit (Kallstrom et al. 2003). In E. coli, over-expression of ObgE rescues the growth and ribosome defects in 
rrmJ strain, a null mutant of rrmJ, rRNA methyltransferase of 23S rRNA (Tan et al. 2002). ObgE over-expression is likely not to restore the 23S rRNA modification but rather stabilize the 70S ribosomes biogenesis in rrmJ strain, thereby overcoming the effects of the absence of methylation. More recently, ObgE was reported to cofractionate with the 50S subunit and interact with SpoT, ppGpp synthetase/hydrolase (Wout et al. 2004). These results raised the question how Obg proteins regulate the ribosomal function.
The E. coli ribosome is composed of a 30S small subunit and a 50S large subunit (Green & Noller 1997) containing 3 species of ribosomal RNAs (rRNAs) (5S, 16S and 23S) and 54 different ribosomal proteins (S1–S21 for 30S, L1–L36 for 50S), and the assembly process is extremely complex (Wada 1986a; Noller & Nomura 1996). Ribosome maturation involves processing and folding of rRNA, modification of both rRNA and ribosomal proteins, and association of ribosomal proteins with rRNA into 70S mature ribosomes. The rRNAs are first transcribed as a 30S precursor transcript containing 16S, 23S, and 5S rRNA sequences. This transcript is cleaved into pre-16S, pre-23S and pre-5S rRNA by RNaseIII, and further processed into mature 16S, 23S and 5S rRNA by specific nucleases. Pre-16S rRNA has 115 extra residues at the 5' end of 16S rRNA and 33 extra residues at the 3' end. Pre-23S rRNA has 3 or 7 extra residues at the 5' end of 23S rRNA and either 7 or 9 residues at the 3' end (Srivastava & Schlessinger 1990). During the ribosome assembly, many ribosomal proteins are post-translationally modified, including acetylation at the N-terminus of S5 (Wittmann-Liebold & Greuer 1978), S18 (Yaguchi 1975) and L12 (Terhost et al. 1973), methylation of at least ten ribosomal proteins (Chen et al. 1977) and the addition of glutamic acid residues at the C-terminus of S6 (Kang et al. 1989). The rRNAs are also subjected to numerous post-transcriptional modifications that are important for the structure of rRNA in ribosome, including pseudouridines substitution and methylations (Decatur & Fournier 2002). Although in vitro reconstitution experiments have shown that ribosome subunits self-assemble, this reaction requires a high temperature, high ionic condition and long incubation compared to the in vivo conditions (Traub & Nomura 1968; Nierhaus 1991), suggesting that additional factors to catalyze the assembly steps might be necessary for construction of functional 70S ribosomes in vivo.
We here demonstrate that ObgE may be such a factor that promotes efficient maturation of ribosomal components in E. coli. ObgE is shown to be associated with 16S and 23S rRNAs, and several specific ribosomal proteins. Our data suggest that ObgE plays a role in biogenesis not only in the 50S subunit but also in the 30S subunit. These results indicate that ObgE is necessary for ribosome maturation perhaps through the binding to the rRNA and/or the rRNA-protein complexes.
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Results |
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The ObgE protein is essential to E. coli cell growth (Kobayashi et al. 2001). To investigate the effect of ObgE on growth, GN5012 strain (KY1416 obgE::kan/pFE215 PT5-lacOid-obgE+) was constructed in which the host chromosomal obgE gene was deleted, and instead, the wild-type ObgE protein was supplied by obgE+ gene directed by an IPTG inducible promoter on pFE215. Plasmid pFE215 contains mini-F replicon (ori2, repE, incC), and thus the copy number is controlled to maintain 1 or 2 copy per cell. In GN5012, the homogeneous expression of ObgE can be induced by isopropyl-1-thio-ß-D-galactopyranoside (IPTG), which can freely penetrate the cell membrane (Khlebnikov & Keasling 2002). This strain grew at almost the same rate as wild-type KY1416 cells in LB containing 0.1 mM IPTG at 37 °C (Fig. 1A) but could not grow in LB without IPTG (data not shown). Overnight cultures of GN5012 grown in LB medium with various concentrations of IPTG were inoculated into the same medium and cultured with shaking at 37 °C. Growth was measured by cell turbidity, and ObgE protein concentration was measured by Western blotting with anti-ObgE. The ObgE concentration was correlated with the IPTG concentration used, and the growth rate was correlated with the cellular levels of ObgE (Fig. 1A,B). These data suggest that ObgE protein is a major growth determinant in E. coli. In the wild-type cells (KY1416 or MG1655 (data not shown)), ObgE proteins were relatively abundant in log phase and gradually decreased toward stationary phase (Fig. 1C) in agreement with the previous results (Kobayashi et al. 2001). This expression pattern is reminiscent of the change in ribosomal proteins whose synthesis rate is well correlated with cell growth rate (Gourse et al. 1996).
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The cellular localization of the ObgE protein was examined by immunofluorescence microscopy using anti-ObgE. Wild-type MG1655 cells grown in LB medium at 37 °C were DAPI-stained for nucleoid and immuno-stained for ObgE and observed by phase contrast and immunofluorescence microscopy (Hiraga et al. 2000). The anti-ObgE signal was mainly detected in the cytosol but not in the nucleoid (Fig. 2), similar to that of ribosomal protein L7/L12 (Azam et al. 2000). This suggested that ObgE functions primarily as a cytoplasmic protein. Although the ObgE signal was strongly detected in log phase cells, the signal was weakly detected in stationary phase cells and in either log or stationary phase cells grown in minimal medium (data not shown), suggesting that ObgE proteins are actively synthesized during log phase in LB medium. These observations are consistent with the growth rate data presented above.
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First, the interaction between ObgE and ribosomal subunits was examined. The wild-type MG1655 cells were grown at 30 °C, harvested at the log phase, and the crude lysates were fractionated on a 5–20% sucrose density gradient. Each fraction was assayed for ribosomes by absorbance at 260 nm and for ObgE by Western blotting. ObgE was cofractionated with both 30S and 50S subunits but not with 70S ribosomes (Fig. 3). ObgE was more abundant in the 50S fraction compared to the 30S fraction. Some free ObgE proteins were detected in the top fractions. This result indicated that ObgE proteins interact with both 30S and 50S subunits. We did not observe any association of ObgE with 70S under the condition employed.
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Next, we examined whether ObgE interacts with RNA. ObgE protein and rRNA were purified as described in Experimental procedures. In SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining, some population of the purified ObgE protein migrated slower (55 kDa) or migrated as a dimer (around 110 kDa) in addition to the expected monomer size (50 kDa). A sulfhydryl (–SH) group reducing reagent (ß-mercaptoethanol or DTT; dithiothreitol) in SDS-PAGE sample buffer abolished the 55 kDa and 110 kDa bands (Fig. 4A). This observation suggests that the ObgE protein containing its S-S bond(s) structure probably migrates to 55 kDa while the ObgE protein in the denatured form migrates to 50 kDa, and that S-S bond is important for the ObgE protein conformation and its dimerization. The purified ObgE protein and rRNA were incubated in the presence or the absence of guanine nucleotides for 30 min at 37 °C. Each of the samples was fractionated by a 15–40% sucrose density gradient centrifugation, and assayed for ObgE by SDS-PAGE followed by Western blotting, and assayed for rRNA by formaldehyde agarose gel electrophoresis with ethidium bromide (EtBr) staining. Significant portions of ObgE cosedimented with 16S and 23S rRNAs in the presence, but not the absence, of GTP (Fig. 4C,E). Furthermore, neither GDP nor ObgE alone failed to give significant cosedimentation (Fig. 4B,D). These results indicated that GTP is required for the cosedimentation of ObgE with 16S and 23S rRNAs under the conditions employed. It should also be noted that the ObgE that cosedimented with rRNAs is mainly in the dimer form (Fig. 4E). In the top fraction, not only monomer form but also dimer and possibly tetramer forms of ObgE were detected. The ObgE in the dimer form increased in presence of guanine nucleotides (see top fraction in Fig. 4B,D,E compared to 4C), and the dimer form ObgE was more abundant in the presence of GTP compared to that in the presence of GDP, suggesting that GTP rather than GDP facilitates the ObgE dimerization. We also found that magnesium was necessary for this dimerization (data not shown). These results suggest that ObgE binds to both 16S and 23S rRNAs possibly in the dimer form in the presence of GTP and magnesium.
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To further analyze the functional targets of ObgE, we have identified ObgE-interacting proteins by pull-down assays. The GN5021 cells expressing the (His)6-tagged ObgE and GN5020 cells expressing (His)6-tag peptide only (control) were grown at 37 °C. The (His)6-tagged ObgE and (His)6-tag control were expressed under the IPTG induction and affinity-purified by Ni-NTA column. Each final eluate was separated by SDS-PAGE and stained by CBB, and all visible protein bands were excised from the gel and identified by MALDI-TOF mass spectrometry (MS) (Fig. 5A). The copurifying proteins in GN5021 were compared to those in GN5020, and the proteins which specifically interacted with ObgE were identified (Fig. 5B). Several ribosomal proteins of both the 30S and 50S subunits (S3, S4, S5, S13, S16, L2, L4, L16 and L17) as well as the RNA helicase CsdA, ClpA chaperone, hypothetical protein 274#5, the RNA polymerase ß and ß' subunit were identified as putative ObgE-associated proteins. 274#5 is a hypothetical protein that has a transmembrane domain. These proteins showed a reproducible and presumably stable interaction, whereas DnaK, S7, S14 and L13 were detected only once in four independent experiments, implying that they may be weakly interacting proteins.
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To investigate the manner in which ObgE affects ribosomal components, we isolated a temperature sensitive mutant of ObgE, denoted as ObgEts (GN5010). This strain has a point mutation (S314P) in the conserved GTPase G5 motif (Kobayashi et al. 2001) and so the product of the obgEts gene is predicted to be defective in the GTPase activity. This strain can grow at 30 °C, but shows a severe growth defect at 42 °C. The crude ribosome fractions derived from the wild-type (MG1655) and ObgEts (GN5010) cells grown at 42 °C were analyzed by RFHR 2D-PAGE (Wada 1986a). We quantified all the ribosomal proteins that were stained with CBB on the 2D-PAGE as described in Experimental procedures. Despite the overall greater signal in the ObgEts cells (Fig. 6B compared to 6A), several r-protein levels showed a marked reduction: the S1, S14, S21 and L10 ribosomal proteins levels decreased 75%, 26%, 71% and 41%, respectively, in the mutant compared to the wild-type cells. Moreover, the S6 and S18 ribosomal proteins displayed a different mobility in the ObgEts cells (Fig. 6A,B). S6 is post-translationally modified by addition of glutamic acid residues to the C-terminus (Kang et al. 1989), whereas S18 is acetylated at alanine residues in the N-terminus (Yaguchi 1975). An acidic shift (S6*) of the S6 spot in the mutant was confirmed by MALDI-TOF MS, suggesting extensive glutamylation at the C-terminus (Fig. 6A,B). MALDI-TOF MS analysis also indicated that the S18 in the ObgEts cells was not fully acetylated, consistent with the reduced molecular weight of S18* (–42 Da) (Fig. 6B).
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We analyzed the effect of obgEts mutation on rRNA processing. Total RNAs were prepared from the wild-type (MG1655) and ObgEts (GN5010) cells grown at 30 °C or 42 °C and were analyzed by formaldehyde agarose gel electrophoresis and EtBr staining. As seen in Fig. 7A, a band slightly larger than 16S rRNA (pre-16S) was specifically enhanced in the ObgEts cells grown at 42 °C. This band was rarely observed in the wild-type cells at 30 °C or 42 °C, or in the ObgEts cells under permissive conditions. To determine whether the band is pre-16S rRNA (17S rRNA) (Young & Steitz 1978), we carried out Northern blotting analysis using two probes (Fig. 7B) against each end (5'- and 3'-) of 17S rRNA. Processing of both ends of the 17S pre-rRNA was remarkably inhibited under ObgE defective condition (ObgEts at 42 °C) (Fig. 7C). The wild-type (MG1655) cells treated with chloramphenicol (CM), which is known to block rRNA processing (Young & Steitz 1978), were used as a control (Fig. 7C). Processing of 17S pre-rRNA was partially hampered in the ObgEts cells at 30 °C, suggesting that the S314P mutation impairs ObgE function moderately at the permissive temperature. The processing of the 5' end was more severely inhibited than that of the 3' end by the ObgE mutation.
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To test whether ObgE is required for 70S ribosome maturation, we analyzed ribosome profiles of the wild-type (MG1655) and ObgEts cells. Overnight cultures were diluted 100-fold with the same medium, grown to Klett units 20 at 30 °C, and then shifted to 42 °C or kept at 30 °C for 1 or 2 h. Each cell lysate was analyzed by 5–20% sucrose density gradient fractionation. The temperature shift drastically reduced 70S ribosome levels in ObgEts cells while free 30S and 50S subunits levels increased, indicating a defect in 70S ribosome formation (Fig. 8). In the ObgEts cells at 42 °C, the 30S peak decreased more quickly than the 50S peak, resulting in aberrant 30S:50S ratio. We collected each fraction corresponding to 30S, 50S and 70S peak and analyzed the ribosomal proteins by RFHR 2D-PAGE. Unexpectedly, a portion of the 30S ribosomal proteins were present in the 50S peak of ObgEts cell, suggesting that 30S homodimers form and sediment with the 50S peak. It should be noted that the reduced 70S ribosome levels as well as increased free 30S and 50S subunits were observed in ObgEts cells even at 30 °C, suggesting that the S314P mutation affects ribosome biogenesis moderately, consistent with the partially inhibited pre-16S rRNA processing at this temperature.
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Discussion |
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In this report, we showed that ObgE possesses 16S and 23S rRNA binding activity, and associates with both 30S and 50S subunits. ObgE interacted with specific 30S and 50S ribosomal proteins, CsdA RNA helicase and the DnaK, ClpA molecular chaperones. Moreover, ObgEts mutant cells showed multiple defects in ribosomal proteins and rRNA, resulting in the reduced amount of 70S ribosome levels. These data suggest that ObgE is a trans-acting factor functioning in both 30S and 50S maturation probably through its binding to rRNAs or rRNA-protein complexes.
ObgE association with ribosomal subunits
The sucrose density gradient centrifugation analysis showed the ObgE association with both the 30S and 50S fractions but not 70S fractions. In addition, the pull-down assay indicated that ObgE interacted with both 30S and 50S ribosomal proteins. These data suggest that ObgE plays a role in the maturation of both the 30S and 50S subunits. More ObgE proteins were associated with the 50S fraction than the 30S fraction in sucrose density gradients, indicating that ObgE has a stronger affinity and/or more binding sites for the 50S than the 30S subunit. Previous studies showed that both ObgE and CgtAc, the Obg homolog in C. crescentus, bind only to the 50S subunits by sucrose density fractionation, and the concentration of NH4Cl in buffer is important for the binding (Lin et al. 2004; Wout et al. 2004). To examine the effect of buffer on ObgE–ribosome subunits interaction, we performed sucrose density gradient centrifugation with the buffer used by Lin et al. (2004), and observed the same association pattern with our data: ObgE was associated with both 30S and 50S subunits but more abundant in the 50S fraction than the 30S fraction. Relatively weak affinity of ObgE to the 30S subunit might result in the failure to detect the ObgE–30S subunits interaction in the previous reports.
The role of ObgE in the rRNA maturation
ObgE cofractionated with both 16S and 23S rRNA in the presence of GTP, suggesting that ObgE binds to both 16S and 23S rRNAs in the GTP form. The rRNA maturation takes place concomitant with the transcription and assembly of ribosomal proteins, indicating that the correct rRNA conformation and its interaction with ribosomal proteins are important for the rRNA processing. ObgE in the GTP form may bind to rRNA possibly to promote rRNA folding and proper assembly of rRNA with ribosomal proteins or stabilize the structure of rRNAs and/or rRNA-ribosomal protein complexes in the ribosome maturation process. Recently it was reported that guanine nucleotides stabilize the binding of Obg to ribosomes in Bacillus subtilis, also consistent with our hypothesis (Zhang & Haldenwang 2004).
Upon the ObgE depletion, processing of both 5'- and 3' ends of pre-16S (17S) rRNA was severely inhibited. In view of the observation that ObgE showed more affinity to 50S subunit than to 30S (Fig. 3) and cosedimented with 23S rRNA, it is possible that ObgE mutation also affects pre-23S rRNA processing in the 50S subunit. It is significantly important to examine the effect of ObgE on pre-23S rRNA processing. It has still been controversial where pre-16S processing occurs in the ribosome biogenesis. Some previous studies suggested that the pre16S processing occurs in the nascent 70S ribosome (Srivastava & Schlessinger 1990; Charollais et al. 2003), while other studies suggested it occurs before the 30S subunit incorporated into the 70S ribosomes (Lindahl 1975; Nierhaus 1991; Xia et al. 2003). Since none of the RNases which is known to cleave pre-16S rRNA was found in the ObgE-associated protein complex in the pull-down assay, we think that ObgE is not directly involved in the enzymatic reaction for rRNA cleavage but rather involved in refolding or stabilizing rRNA/ribosomal protein structure. We suppose that the ObgE mutation affects rRNA or rRNA/ribosomal protein structure, resulted in the inhibition of proper pre16S rRNA processing.
It was observed that ObgE protein multimerize both in vivo and in vitro by SDS-PAGE and Native-PAGE in the absence of DTT or ß-mercaptoethanol in the sample loading buffer (Fig. 4 and data not shown). S-S bond is likely to be important to the ObgE multimerlization. The previous study which suggested that ObgE is eluted as a stable multimer form in gel filtration chromatography (Wout et al. 2004) also supports our observation. The GTPase Era, whose DNA sequence is most similar to ObgE among the GTP binding proteins and contains RNA binding domain, binds to 16S rRNA (Meier et al. 1999) and is also crystallized in the dimer form (Chen et al. 1999). These GTPases might interact with rRNAs in the dimer or multimer form and promote rRNA folding in the premature ribosome complex. Because the amino acid sequence of ObgE protein does not contain any previously identified RNA binding motifs, it is a great interest to identify a novel motif to be necessary for rRNA binding.
The nature of ObgE associated complex
ObgE interacted with several ribosomal proteins (Fig. 5), which include five rRNA binding proteins S4, S7, L2, L4 and L17, and other proteins S16, S13, S5, S3 and L16, whose binding is mediated by S4, S7, L2, L4 or L17. These interactions are reproducible in pull-down assays, and this specificity suggests that there is a specific region which ObgE binds in the rRNA or rRNA-protein complex. ObgE also interacted with ribosome-related proteins like CsdA, a DEAD-box helicase, which was isolated as a multicopy suppressor of an rpsB mutation (Toone et al. 1991; Moll et al. 2002). Recently Charollais et al. (2004) showed that CsdA associates with the 50S subunit to help 50S subunit maturation at 20 °C. Though CsdA in ribosome subunits was not observed at 37 °C by Charollais et al. (2004), a small amount of CsdA expressed at this temperature might assist 50S subunit maturation together with ObgE. DnaK and ClpA chaperones were detected as ObgE-interacting proteins. In E. coli, thermosensitive DnaK mutants showed ribosome assembly defects at a non-permissive temperature (Alix & Guerin 1993) and comigration of DnaK with the 50S ribosome subunit was reported (Vysokanov 1995), though DnaK function on the ribosome assembly is still controversial (Alix & Nierhaus 2003). ClpA is a molecular chaperone to facilitate the refolding of DNA replication initiator protein RepA (Pak & Wickner 1997). The interaction of ObgE with DnaK and ClpA might suggest their potential role as chaperones in the ribosome maturation, or might be due to the effect of over-expression of (His)6-tagged ObgE. The RNA polymerase core subunits ß and ß' also showed a specific association with (His)6-tagged ObgE. In the ribosome biogenesis, rRNA processing and assembly with ribosomal proteins are coupled to the synthesis of rRNA (Lewicki et al. 1993). ObgE may interact with nascent rRNAs that still have transcribing RNA polymerase at the 3' end. Alternatively, ObgE might interact directly with RNA polymerase by some unknown mechanism. 274#5 is a hypothetical protein which has a transmembrane domain, and it is now under investigation how this protein is related to ObgE. Each ObgE-associated protein in the pull-down assay was tested for being a multicopy suppressor of GN5010 (ObgEts) cells as described in Experimental procedures, but none of them was capable of rescuing the growth defect of GN5010 (ObgEts) at the non-permissive temperature (data not shown). Although the protein levels of S1, S14, S21 and L10 were significantly reduced and the post-translational modifications of S6 and S18 were altered in the obgE mutants, interactions between these proteins and ObgE were not detected in the pull-down assays. Even though ObgE did not interact with these proteins directly, the defects caused by the ObgE deficiency might impair the proper assembly of the 30S and 50S subunit components and subsequently impair the modifications and ribosomal protein levels. Because ribosome assembly and pre-rRNA processing are intimately linked and cooperative processes, one defect can be the cause or the consequence of the other. Since we found that ObgE binds to rRNA directly, we propose that ObgE mutation primarily affects the structure of rRNAs and/or rRNA-ribosomal protein complexes, and this consequently hampered rRNA processing and causes multiple defects in ribosome maturation process.
It has been reported that the Obg protein in the GTP form prevents cellular differentiation in S. coelicolor and B. subtilis (Welsh et al. 1994; Okamoto & Ochi 1998). Obg in B. subtilis is also reported to crystallize with guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a signal molecule for amino acid starvation (Buglino et al. 2002). These reports brought us the proposal that Obg functions as a sensor of intracellular GTP levels. We suggest that Obg in the GTP-bound form participates in the ribosome biogenesis and consequently promotes the protein synthesis in the nutritionally rich environment in which cellular GTP pool is elevated while its GDP-bound form or ppGpp-bound form plays a role in the cellular differentiation or stringent response, respectively. Further investigation is needed to address the question whether the energy of GTP hydrolysis is used to structurally rearrange the pre-rRNA or pre-rRNA-protein complex, or used to catalyze the pre-rRNA processing reactions.
Though pleiotropic phenotypes were reported in obg mutants in various species, our data here indicate that ObgE functions in ribosome maturation for both 30S and 50S subunits. Previously we reported that ObgE is essential for chromosome partitioning (Kobayashi et al. 2001). It is most likely that the impaired ribosome function in ObgE defective conditions, resulting in the inhibited protein synthesis, causes the impeded chromosome partition indirectly, though we cannot exclude the possibility that ObgE also concerns directly chromosome partition by some unknown mechanism.
In summary, here we propose that ObgE is required for both 30S and 50S subunit maturation and assembly process through the binding to rRNAs and/or RNA-protein complexes.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The bacterial strains of E. coli K-12 and plasmids used in this study are listed in Table 1. Plasmid pFE215-obgE+ was constructed as follows. First, gfp and hexa-histidine genes were removed from plasmid pCA24N (Mori et al. 2000) by NotI digestion and PCR-ligation-PCR method (Ali & Steinkasserer 1995), respectively, and the DraI-SapI fragment containing lacIq, PT5-lacOid and TrrnB was inserted into a unique XhoI site of mini-F pKV713 (Uga et al. 1999), generating pFE100. Plasmid pFE100 contains lacIq, PT5-lacOid and TrrnB as well as bla gene and ori2 derived from pKV713. Furthermore, pFE100 was modified by inverse PCR and self-ligation to generate a start codon (ATG) in the upstream of SfiI site, and this plasmid is called pFE215 (Natsuko Yamamoto, unpublished data). Strain GN5012 was constructed by obgE::kan P1 transduction to KY1461(HI2017 thy+ F–: (ara-leu)697, (lac-pro), thi, trpA38(OC)) carrying pFE215-obgE+, in which the obgE+ gene is inserted into the SfiI site of pFE215 and thus expressed by lac promoter upon induction with IPTG. The obgE temperature sensitive mutant strain GN5010 was constructed as follows. First, strain GN5002 was constructed by obgE::kan P1 transduction to MG1655 cells that carry pGK14 (ori-pSC101cat PBAD-obgE+) in which the obgE+ gene is expressed by the pBAD promoter upon induction with L-arabinose. Next, the temperature sensitive mutants from GN5002 (MG1655obgE::kan/pGK14(PBAD-obgE+)) were isolated by PCR mutagenesis of the obgE gene on pGK14. A useful mutant carried two mutations (S314P and N335D). A point mutation, which converts the 314 serine residue of ObgE to praline, showed temperature sensitivity and was introduced in the obgE gene of pGK14 using the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). This plasmid was called pGK16 (ori-pSC101cat PBAD-obgEts). Finally, GN5001 (MG1655 obgE::kan[pGK13(ori-pSC101bla reptsPBAD-obgE+)] cells were transformed with pGK16, and then a chloramphenicol resistant and ampicillin sensitive cell was isolated at 30 °C and denoted as GN5010 (ObgEts). The construction was confirmed by DNA sequencing. GN5010 showed normal growth rates at 30 °C in LB medium with 0.003%L-arabinose but hardly grew at 42 °C (Fig. 8A). The ser314 in the obgE gene is a conserved residue in the G5 domain (V/ISAX) of the GTP binding motif (Bourne et al. 1991; Kobayashi et al. 2001), and so the product of the obgEts gene is predicted to be defective in the GTPase activity. For pull-down assay, the obgE gene fragment derived from BamHI and HindIII digestion of pGK2 was inserted into corresponding sites of pQE9, which generated pQE9-obgE+ plasmid. The control strain GN5020 was constructed by transforming MC4100 with pQE9 and pREP4 while GN5021 was constructed by transforming MC4100 with pQE9-obgE+ and pREP4.
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In most cases, bacteria were grown in the modified LB medium (1% Bacto Tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.4) in a water-bath shaker or on LB plates containing 1.2% agar. M-9 was used as a minimal medium. Unless otherwise specified, GN5010 cells were grown in LB medium with 0.003%L-arabinose (Nacalai tesque, Kyoto, Japan). In the condition, the expression level of ObgE is the same level as the wild-type strain. When necessary, selected media containing: kanamycin (25 µg/mL), chloramphenicol (20 µg/mL) and ampicillin (50 µg/mL) were used. Cell growth in liquid media was followed by measuring turbidity with a Klett Summerson photoelectric colorimeter (KLETT MFG. Co. Inc., New York, USA) with green filter (#54), or a Shimadzu photoelectric colorimeter UV-180 OD600 (Shimadzu, Kyoto, Japan). Turbidity was expressed in Klett Units or OD600.
Western blotting
Unless otherwise specified, Western blotting was performed as follows. Samples were mixed with 2x sample buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 2 mM dithiothreitol (DTT)), subjected to 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immuno-Blot polyvinylidene difluoride (PVDF) membrane (Bio-rad, Hercules, CA, USA) with semidry transfer apparatus (Bio-rad). The ObgE proteins were probed with anti-ObgE and Horse radish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and visualized with ECL chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer's instruction.
Immuno-fluorescence microscopy
The polyclonal anti-ObgE antibody was precleared to exclude the nonspecific cross-react proteins using a PVDF membrane to which E. coli lysate was transferred as follows. MG1655 cell lysate was subjected to 12% SDS-PAGE and transferred to PVDF membrane. The part of membrane corresponding to ObgE protein (50 kDa on the gel) was cut out, and the remaining PVDF membrane was incubated with polyclonal anti-ObgE antibody overnight. The supernatant of antibody solution was used for immuno-fluorescence microscopy analysis as previously described (Hiraga et al. 2000).
Sucrose density gradient centrifugation of ribosomes
Overnight cultured cells (0.2 mL) were inoculated into 40 mL of the fresh medium, and further grown at 30 °C. The cells at log phase were resuspended in 400 µL of buffer I (20 mM Tris-HCl pH 7.6, 15 mM magnesium acetate, 100 mM ammonium acetate), mixed with an equal volume of glass beads (G-1277; Sigma, St Louis, MO, USA), and vortexed six times for 30 s. After centrifugation at 9100 g for 15 min, protein concentration of the supernatant was measured using BCA protein assay kit (Pierce, Rockford, IL, USA). Samples of 0.327 mg total protein were layered on to a 12 mL gradient of 5–20% sucrose in buffer I and centrifuged in an SW40 Ti rotor (Beckman Instruments, Fullerton, CA, USA) at 40 000 r.p.m. for 80 min (Fig. 8B) or 130 min (Fig. 3) at 4 °C. For the Fig. 3 experiment, fractions of 550 µL were collected from the top, and the UV absorbance (260 nm) was measured by a Shimadzu UV180 spectrophotometer. For Western blotting (Fig. 3), proteins in each fraction were precipitated with 10% trichloroacetic acid, resuspended in SDS sample buffer containing 2 mM DTT and separated by 12% SDS-PAGE. The proteins on the gel were transferred to PVDF membrane. The membrane was blocked with 10% skim milk in TBS-T (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20), probed with anti-ObgE and washed with TBS-T. The bound antibody was detected with Alkaline phosphatase conjugated secondary antibody against rabbit IgG (Cappel, Irvine, CA, USA) and visualized using 5-bromo-4-chloro-3-indolyl phosphate/p-nitro blue tetrazolium system (BCIP/NBT) system.
RNA-binding assays
RNA-binding assays with ObgE were carried out using the modified ribosome reconstitution buffer (buffer IV*) (30 mM Tris-HCl pH 7.4, 20 mM MgCl2) (Held et al. 1973). Crude ribosome fraction was prepared as previously described (Wada 1986b), and 16S and 23S rRNAs were extracted from the crude ribosome fractions by phenol-chloroform treatment and dialyzed against buffer IV*. The ObgE proteins, purified as described before (Kobayashi et al. 2001), were dialyzed against buffer IV* containing 0.5 M KCl. ObgE proteins were mixed with purified rRNAs (Fig. 4C–E) in a molar ratio of 1 : 1, or buffer IV* only for control (Fig. 4B). 10 mM GTP or GDP was added, and the mixture was incubated at 37 °C for 30 min, cooled to room temperature, and then applied on to a 15–40% sucrose density gradient in buffer I. The samples were centrifuged at 48 000 r.p.m. for 160 min in a MLS5 rotor (Beckman Instruments). Each ten drops was collected from the bottom to top and suspended in equal volume of SDS-PAGE sample buffer without SH reducing reagent. The samples were subjected to SDS-PAGE followed by ECL-Western blotting with anti-ObgE. The RNAs in each fraction were mixed with RNA sample buffer, incubated at 70 °C for 10 min, and then subjected to 1.0% agarose gel electrophoresis with 1.8% formaldehyde. RNA electrophoresis was carried out by the conventional protocols with ethidium bromide (EtBr) staining, and the RNAs were photographed under the UV light (Sambrook et al. 1989).
Pull-down assays
Affinity purification of the (His)6-tagged ObgE protein was performed using a QIA expression kit (Qiagen), with the following modifications. GN5021 expressing (His)6-tagged ObgE protein and GN5020 expressing only (His)6-tag (control) were grown overnight in a selected LB medium, and 1 mL overnight culture was inoculated in 200 mL of the fresh medium, and further shaken at 37 °C. Isopropylthio1-ß-D-galactoside (IPTG, 0.1 mM) was added at OD600 0.6 and the cells were harvested after 3 h. The cell pellets were frozen, thawed and resuspended in 1.2 mL Lysis buffer (50 mM NaH2PO4, 100 mM NaCl, 20 mM Imidazole, 0.1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-2-hydroxyl-1-propanesulfonate (CHAPS), pH 8.0). Lysozyme and 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) were added, and the cell lysates were incubated on ice for 30 min, and then disrupted with a French press. After centrifugation for 20 min at 15 000 g, the supernatant was mixed with Ni-NTA resin (Qiagen) for 1 h by rotating, then washed with Wash buffer (50 mM NaH2PO4, 200 mM NaCl, Imidazole 50 mM, 0.1% CHAPS, pH 8.0) four times, and eluted with Elution buffer (50 mM NaH2PO4, 8 M Urea, 1.5 M NaCl, pH 8.0). The eluate was concentrated with a Centricon-3 (Millipore, Billerica, MA, USA). Proteins were separated using 10–20% Nu-PAGE gels (Invitrogen, Carlsbad, CA, USA) and visualized by Coomassie Brilliant Blue (CBB) R-250. All proteins visualized by CBB staining in the SDS-PAGE were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (Voyager DE-PRO, Applied Biosystems, Foster City, CA, USA) (Maeda et al. 2003).
Test for multicopy suppressor of an obgEts mutant
Each gene of the protein which interacted with ObgE in the pull-down assay was cloned into pTrc99A (Amersham Biosciences) in which the protein can be over-expressed upon IPTG induction. Each plasmid was used to transform GN5010 (MG1655obgE/PBAD-obgEts) and GN5002 (MG1655obgE/PBAD-obgE+). GN5010 displays a severe growth defect on LB plates containing 0.003% arabinose and chloramphenicol at 42 °C while GN5002 shows a robust growth under the same condition. Both GN5010 and GN5002 strains harboring each plasmid was inoculated into selective LB medium (LB +chloramphenicol +ampicillin +0.003%L-arabinose) at 30 °C overnight. The overnight culture was plated with appropriate dilutions on to the selective LB plate or the selective LB plates containing 1 mM IPTG, and then incubated at 30 °C (control) or 42 °C. Complementation of obgEts phenotype was tested by the ability of the clones to restore the growth defect after overnight incubation at 42 °C on the selective LB plates containing 1 mM IPTG.
RFHR two-dimensional electrophoresis of ribosomal proteins
Cells grown to Klett units 40 at 42 °C in 400 mL LB medium were harvested. Cells were re-suspended in buffer I and sonicated, and the crude ribosome fraction was prepared as described before (Wada 1986b). Nucleic acid in the crude ribosome fraction was eliminated by the acetic acid method (Hardy et al. 1969). Briefly, 1/30 volume of 1 M MgCl2 and 2 volumes of acetic acid were added and the mixture was vortexed for 1 h at 4 °C. After centrifugation at 10 000 g for 10 min, the supernatant was desalted by Sephadex G-25 (Medium) column (Amersham Pharmacia Biotech), the desalted supernatant was lyophilized. Protein samples of 1 mg were analyzed by Radical Free and Highly Reducing two-dimensional-PAGE (RFHR 2-D) (Wada 1986a; Oshima et al. 2002). Gels were stained with CBB and identified by MALDI-TOF MS. All the protein spots on the 2D-PAGE were quantified by Scion Image Beta 4.02 software (Scion Corporation), and a relative abundance of each ribosomal protein in the total protein mass was compared between wild-type (MG1655) and ObgEts (GN5010) cells.
Northern blot analysis
The analysis was performed essentially as previously described (Sambrook et al. 1989) using specific probes shown in Fig. 7B. The total RNA from wild-type (MG1655) and ObgEts (GN5010) cells were prepared by the hot phenol method (Aiba 1985). The RNAs were mixed with an equal volume of formamide, denatured by heating at 65 °C for 15 min, separated by 1.0% agarose gel electrophoresis with 2% formamide and transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech). Northern blotting analysis was performed using an AlkPhos direct system (Amersham Pharmacia Biotech), according to the manufacturer's instructions with both probes (5'-GACGTTAAGGAATCCGTATCTTCGAGTGCCCACA-3') for 5'+115 and (5'-TGTGTGAGCACTACAAAGTACGCTTCTTTAAGG-3') for 3'+33.
Other general methods
DNA manipulations and SDS-PAGE were performed by standard procedures (Sambrook et al. 1989).
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Acknowledgements |
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Footnotes |
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aPresent address: Research and Education Center of Informatics, Nara Institute of Science and Technology, Nara 630-0101, Japan.
* Correspondence: E-mail: cwada{at}lif.kyoto-u.ac.jp |
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Received: 11 January 2005
Accepted: 24 January 2005
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 Y. Hirano, R. L. Ohniwa, C. Wada, S. H. Yoshimura, and K. Takeyasu Human small G proteins, ObgH1, and ObgH2, participate in the maintenance of mitochondria and nucleolar architectures Genes Cells, November 1, 2006; 11(11): 1295 - 1304. [Abstract] [Full Text] [PDF]
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M. Jiang, K. Datta, A. Walker, J. Strahler, P. Bagamasbad, P. C. Andrews, and J. R. Maddock The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly. J. Bacteriol., October 1, 2006; 188(19): 6757 - 6770. [Abstract] [Full Text] [PDF]
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A. E. Sikora, R. Zielke, K. Datta, and J. R. Maddock The Vibrio harveyi GTPase CgtAV Is Essential and Is Associated with the 50S Ribosomal Subunit J. Bacteriol., February 1, 2006; 188(3): 1205 - 1210. [Abstract] [Full Text] [PDF]
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