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Genes to Cells (2009) 14, 271-280. doi:10.1111/j.1365-2443.2008.01272.x
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Activities of Escherichia coli ribosomes in IF3 and RMF change to prepare 100S ribosome formation on entering the stationary growth phase

 Hideji Yoshida1a,*, Masami Ueta1,2, Yasushi Maki1, Akiko Sakai3 and Akira Wada1,2

1 Department of Physics, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
2 Yoshida Biological Laboratory, Yamashina, Kyoto 607-8081, Japan
3 Department of Chemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The canonical ribosome cycle in bacteria consists of initiation, elongation, termination, and recycling stages. After the recycling stage, initiation factor 3 (IF3) stabilizes ribosomal dissociation by binding to 30S subunits for the next round of translation. On the other hand, during the stationary growth phase, it has been elucidated that Escherichia coli ribosomes are dimerized (100S ribosome formation) by binding ribosome modulation factor (RMF) and hibernation promoting factor (HPF), leading to a hibernation stage. This indicates that 100S ribosomes are formed after these factors are scrambled for ribosomes concomitantly with transition from the log phase to the stationary phase. In this study, to elucidate the ribosomal events before 100S ribosome formation, the relationships between protein factors (RMF and HPF) involved in 100S ribosome formation and IF3 involved in initiation complex formation were examined. As a result of in vitro assays, it was found that ribosomal dissociation activity by IF3 fell, and that ribosomal dimerization activity by RMF and HPF was elevated more when using stationary-phase ribosomes than when using log-phase ribosomes. This suggests that ribosomes change into forms which are hard to bind with IF3 and easy to form 100S ribosomes by RMF and HPF concomitantly with transition from the log phase to the stationary phase.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
One of the most important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. Ribosomes are universally conserved ribonucleoproteins and are comprised of two asymmetric subunits. In bacteria, large (50S) and small (30S) subunits associate to form functional 70S ribosomes. Ribosomes can account for as much as 45% of the total mass of bacterial cells, such as Escherichia coli, during the logarithmic (log) growth phase, which can actively synthesize various proteins; however, in cells under stress conditions such as starvation, ribosomal biosynthesis is repressed and protein synthesis is also suppressed. These systems enabling translational regulation are very important for wild bacteria surviving harsh environments.

In eukaryotes, it is known that the phosphorylation of initiation factor-2 {alpha} (eIF2 {alpha}) is an adaptive mechanism for down-regulating protein synthesis under stress conditions (Hinnebusch 1994). On one hand, in the proteobacteria gamma group, including E. coli, protein synthesis is mainly suppressed by the formation of 100S ribosomes (Wada et al. 1990). The 100S ribosome is a dimer of 70S ribosomes, which is formed by ribosome modulation factor (RMF) binding to the ribosomes. The RMF is a basic (pI = 11.3) and small (Mr = 6507) protein, and its expression remarkably increases during transition from the log to stationary phase. Another protein factor expressed during the stationary phase, hibernation promoting factor (HPF), also binds to ribosomes and promotes 100S ribosome formation by RMF (Maki et al. 2000; Ueta et al. 2005). The 100S ribosomes have no translational activity and are formed during the stationary phase (Wada et al. 1995). In previous studies, it has been elucidated that RMF inactivates ribosomes by covering the peptidyl transferase center and the entrance to the peptide exit tunnel (Yoshida et al. 2002, 2004). When stationary-phase cells are transferred to rich nutritious culture medium, RMF is immediately released from 100S ribosomes, which dissociate back into 70S ribosomes (Wada 1998). This process is rapid and is completed within 1 min (Aiso et al. 2005). After this process, cells can reinitiate protein synthesis and proliferation within 6 min. The E. coli strain with deleted rmf gene cannot form 100S ribosomes and its lifetime is shorter than that of the wild strain (Yamagishi et al. 1993). These phenomena indicate that an interconversion system between 70S and 100S ribosomes is an important strategy for survival under stress conditions. The ribosomal resting stage, namely the stage of 100S ribosome formation, is incorporated into a ribosome cycle and is called the hibernation stage (Yoshida et al. 2002).

The canonical ribosome cycle in bacteria consists of initiation, elongation, termination, and recycling stages. During the log phase, ribosomes in the recycling stage are dissociated into subunits by ribosome recycling factor and elongation factor-G (EF-G) in a GTP-dependent reaction, and then IF3 stabilizes the dissociation by binding to 30S subunits for the next round of translation (Karimi et al. 1999; Hirokawa et al. 2005). On the other hand, it has been elucidated that most ribosomes during the stationary phase are dimerized (100S ribosome formation) by binding RMF and HPF, leading to the hibernation stage (Yoshida et al. 2004). This indicates that 100S ribosomes are formed after these factors (IF3 and RMF–HPF) are scrambled for ribosomes concomitantly with transition from the log phase to the stationary phase. What events occur before ribosomal dimerization? Do the amounts of IF3 and RMF in cells decrease and increase concomitantly by entering the stationary phase, respectively? Or do free 70S ribosomes which RMF can bind after the ribosomal recycling process increase during the stationary phase even if IF3 exists? Or can RMF exclude IF3 from 30S subunits to form 100S ribosomes? In this study, to answer these questions, the relationships between protein factors (RMF and HPF) involved in 100S ribosome formation and IF3 involved in initiation complex formation were examined. These factors were added to ribosomes extracted from E. coli cells of the log or stationary phase in vitro. As a result, it was elucidated that ribosomal dissociation activity by IF3 was higher when using log-phase ribosomes than stationary-phase ribosomes because IF3 was difficult to bind to stationary-phase ribosomes. In contrast, ribosomal dimerization (100S ribosome formation) activity by RMF and HPF was higher when using stationary-phase ribosomes than log-phase ribosomes, although there was no significant difference in the binding activity of RMF. This indicates that ribosomes change into those which are difficult to bind with IF3 and easily form 100S ribosomes by RMF and HPF concomitantly with transition from the log to stationary phase.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Quantitative difference in IF3 between log and stationary phases

During the log phase, ribosomal dissociation into subunits is stabilized by IF3 after the recycling process. During the stationary phase, however, most ribosomes are converted to 100S ribosomes by RMF and HPF. This means that IF3 binds to ribosomes after the recycling process during the log phase, but RMF and HPF bind to ribosomes during the stationary phase. This phenomenon can be explained by assuming that IF3 in the cell decreases and RMF and HPF increase concomitantly with entering the stationary phase. In a previous study, it was elucidated that RMF and HPF are exclusively expressed during the stationary phase (Wada et al. 1990; Maki et al. 2000). On the other hand, it has been reported that the expression of IF3 depends on the growth rate and the molar ratio of IF3 to ribosomes (IF3/ribosomes) is kept constant at about 0.2 (Howe & Hershey 1983). However, there are also reports that the expressions of IFs and the IF : ribosome ratio vary under cold shock stress in which the growth rate is low (Giuliodori et al. 2004, 2007); therefore, it is unknown whether the amount of IF3 is different between the log and stationary phases. To answer this question, the amount of IF3 during both phases was examined by RFHR 2-D PAGE, as shown in Fig. 1 (Wada 1986). Some ribosomal proteins on the gels were unstable when total proteins were analyzed by 2-D PAGE; however, protein factors bound to ribosomes such as IF3 and RMF could be stably detected. Although the spot of HPF was not found due to interference with non-ribosomal protein spots in the acidic region of the gels, HPF has been identified as a stationary-phase specific protein in previous studies (Maki et al. 2000; Ueta et al. 2005). Stationary-phase-specific proteins RMF and SRA could be detected during the stationary phase, as shown in Fig. 1B, but not during the log phase, in Fig. 1A. This shows that stationary-phase-specific proteins derived from cells before inoculation did not remain in the log-phase cells examined in this work, and stationary-phase-specific proteins were not expressed. As a result, Fig. 1A,B show that IF3/ribosomes during the stationary phase were almost the same (about 0.2) as during the log phase, although ribosomes/cell and IF3/cell during the stationary phase decreased in comparison with those during the log phase. This was confirmed by more than five repeats of experiments, indicating that both IF3 and RMF exist in stationary-phase cells.


Figure 1
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  		Figure 1  Quantitative differences of IF3 between log and stationary phases. Total proteins extracted from E. coli cells cultivated for 2.5 h (A, Log phase) and for 4 days (B, Stationary phase) were subjected to RFHR 2-D PAGE. IF3 is indicated in red. RMF and SRA (stationary-phase-specific proteins) are indicated in blue, and most ribosomal proteins are also shown. Molar ratios of IF3 in log and stationary phases were measured by normalization against the average amount of ribosomal protein.

 
Binding of IF3 and RMF to ribosomes in vivo

Figure 2A, the ribosome profile by sucrose density gradient centrifugation (SDGC), shows that the cell extract during the log phase (Log-CE) includes 30S and 50S subunits, and a number of 70S ribosomes. On the other hand, the cell extract during the stationary phase (Stat-CE) includes a number of 100S ribosomes, as shown in Fig. 2B, in addition to the particles included in Log-CE. Although it was shown that IF3 and RMF existed in stationary-phase cells in Fig. 1B, it is unknown whether these proteins bound to ribosomes in vivo. Binding was examined by Western blotting, as shown in the lower columns of Fig. 2. In these experiments, most IF3 existed in the top fraction during both phases; however, it is believed that IF3 is normally bound to 30S subunits during the log phase in which protein synthesis activity is high. This might mean that IF3 transiently binds to ribosomes during the log phase, or IF3 is released from 30S subunits in the translational initiation process in vitro before loading to SDGC. The result of the stationary phase might be reasonable, because it is commonly believed that IF3 does not bind to ribosomes during the stationary phase in which protein synthesis activity is low. On the other hand, RMF is non-existent during the log phase and binds to ribosomes exclusively during the stationary phase.


Figure 2
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  		Figure 2  Binding of IF3 and RMF to ribosomes in vivo. Ribosome profiles in cell extracts during the log phase (A: Log-CE) and stationary phase (B: Stat-CE). Sedimentation is from left to right. Positions of 30S, 50S, 70S, and 100S are indicated. The binding of IF3 and RMF to ribosomes was examined by Western blotting, as shown in lower columns (see Experimental Procedures).

 
Additional effects of IF3 or RMF to log- or stationary-phase ribosomes in vitro

Translation factors and protein factors (RMF and HPF) bind to crude ribosomes. To eliminate these proteins from ribosomes for use in in vitro assays, the crude ribosomes of log and stationary phases were washed with high-salt buffer and treated with low-magnesium-ion (Mg2+) buffer (see Experimental procedures), called Log-HLR and Stat-HLR here (Fig. 3A,D), respectively. The 100S ribosomes disappeared by removing RMF and HPF, as shown in Fig. 3D. tRNA was also eliminated from crude ribosomes, because the ribosomes were dissociated to subunits by treatment using Low-Mg2+ buffer (data not shown). In the following in vitro assays, these HLRs (Fig. 3A,D) were generally used.


Figure 3
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  		Figure 3  Additional effect of IF3 or RMF on log- or stationary-phase ribosomes in vitro. (A)–(H) Sedimentation behaviors of ribosomes alone or mixtures of ribosomes and the protein factor (IF3 or RMF) were analyzed as described in Experimental procedures. Sedimentation is from left to right. High-salt-washed and low-Mg2+-treated ribosomes (HLR, 30 nmol) prepared from Log-CR and Stat-CR were incubated alone (A, D), with IF3 (B, E, IF3/ribosome = 40), and with RMF (C, F, RMF/ribosome = 10) at 37 °C for 30 min. The dependence of activities of IF3 and RMF on molar ratios of these protein factors to ribosomes (HLR) is shown in (G) and (H), respectively. Each activity of IF3 and RMF is presented by normalizing to the maximum heights of 50S and dimer peaks in ribosome profiles, respectively.

 
To measure the ribosomal dissociation activity of IF3 or the ribosomal dimerization activity of RMF against log- or stationary-phase ribosomes, these protein factors were added to Log-HLR or Stat-HLR in vitro, the mixtures were incubated at 37 °C for 30 min, and then ribosome profiles were analyzed by SDGC. When IF3 was added to Log- or Stat-HLR, some of the 70S ribosomes were dissociated to subunits, as shown in Fig. 3B and 3E. Dissociation activity by IF3 was higher when using Log-HLR (Fig. 3B) than Stat-HLR (Fig. 3E). On the other hand, when RMF was added to Log- or Stat-HLR, the dimer (100S ribosome) was observed when using Stat-HLR, as shown in Fig. 3F, but not when using Log-HLR, as shown in Fig. 3C; more specifically, dimerization activity by RMF was higher when using Stat-HLR than Log-HLR. Figure 3G,H show the dependence of IF3 and RMF activities on molar ratios of these factors to ribosomes, respectively. The dissociation activity of IF3 was about two times higher in Log-HLR than Stat-HLR in Fig. 3G. In contrast, the dimerization activity of RMF was about four times higher in Stat-HLR than Log-HLR in Fig. 3H. The molar ratio of IF3 to ribosomes is about 0.2, and that of RMF does not exceed 1.0 in E. coli cells. Accordingly, the amounts of factors used in these assays are in excess. In particular, a significant amount of IF3 was needed. This problem may have been caused because IF3 had a 6 x His-tag at the N-terminus and association buffer suitable for ribosome dimerization was used in these assays. On the other hand, it is known that RMF is easily degraded. This may explain why excess RMF was needed. Although these experiments were performed under such non-physiological conditions, IF3 and RMF activities against Log-HLR and Stat-HLR can be compared because the experimental conditions were the same in both cases. As a result, the magnitude relationships of IF3 and RMF activities against Log-HLR and Stat-HLR were maintained in all repeated experiments.

Binding of IF3 and RMF to ribosomes in vitro

It was examined whether the binding of IF3 and RMF to ribosomes affects the activities of these factors. IF3 or RMF was added to Log-HLR or Stat-HLR and the binding was analyzed by Western blotting, as shown in Fig. 4 (see Experimental procedures). To clarify the binding differences, small amounts of factors were used in these experiments. As can be seen in Fig. 4A, IF3 was included in 30S and 70S fractions for Log-HLR. When a sufficient amount of IF3 was added to ribosomes, most ribosomes were dissociated into subunits and most IF3 bound to 30S subunits, as might be expected (data not shown); however, most ribosomes were not dissociated to subunits under this experimental condition (IF3/ribosome = 10), as shown in Fig. 3G, and most IF3 unexpectedly bound to 70S ribosomes. The binding of IF3 to 70S ribosomes may be artificial because there are reports that IF3 has a tendency to bind non-specifically to 70S ribosomes (Dallas & Noller 2001; Pioletti et al. 2001). In these experiments, the most notable fact is that the binding of IF3 to Stat-HLR was not observed despite the same conditions as for Log-HLR. This indicates that IF3 hardly bound to Stat-HLR in comparison with Log-HLR under this experimental condition.


Figure 4
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  		Figure 4  Binding of IF3 and RMF to ribosomes in vitro. The mixtures of ribosomes and factors were fractionated by SDGC, and the factors contained in each fraction were detected by Western blotting (see Experimental procedures). Sedimentation is from left to right. Positions of 30S, 50S, 70S, and dimer (100S) are indicated by arrows. (A) IF3 was added to Log-HLR and Stat-HLR at a molar ratio of IF3/ribosome = 10, and (B) RMF was added to Log-HLR and Stat-HLR at a molar ratio of RMF/ribosome = 1.

 
On the other hand, there was no significant difference in the amount of RMF bound to ribosomes between Log- and Stat-HLRs, as seen in Fig. 4B. RMF mainly binds to 50S subunits and 70S ribosomes in Log-HLR. In Stat-HLR, RMF also binds to 100S (dimer) ribosomes.

Relationships between IF3 and RMF

It was examined whether IF3 and RMF interfered with each other. First, IF3 (IF3/ribosome = 40) was added to Stat-HLR (ribosome profile is shown in Fig. 3E), and then excess RMF (RMF/ribosome = 20) was added, as shown in Fig. 5A. In comparison with Figs 3E and 5A, the peaks of 30S and 50S subunits were unchanged, that of 70S was lowered, and a slight dimer was formed, as shown by an arrow. In a previous study, it was elucidated that RMF bound to 50S subunits exclusively when 100S ribosomes were dissociated to 30S and 50S subunits by treatment with low Mg2+ buffer (Wada et al. 1990). This indicates that IF3 must be released from 30S subunits before 100S ribosome formation even if RMF can bind to 50S subunits.


Figure 5
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  		Figure 5  Relationships between IF3 and RMF. Mixtures of IF3, RMF and ribosomes were analyzed by SDGC. Sedimentation is from left to right. (A) IF3 (IF3/ribosome = 40) was added to Stat-HLR, and then excess RMF (RMF/ribosome = 20) was added. Arrow shows the dimer formed by the addition of RMF. (B) RMF (RMF/ribosome = 10) was added to Stat-HLR, and then excess IF3 (IF3/ribosome = 80) was added. These results should be compared with Fig. 3E,F.

 
Next, RMF (RMF/ribosome = 10) was added to Stat-HLR (ribosome profile is shown in Fig. 3F), and then excess IF3 (IF3/ribosome = 80) was added, as shown in Fig. 5B. In comparison with .Figs 3F and 5B, the dimer disappeared with the addition of excess IF3. Although the experimental condition is non-physiological, this result might indicate that IF3 acts to dissociate 100S ribosomes to 70S ribosomes.

Additional effects of HPF

Dimer formation activity when using stationary-phase ribosomes is higher than when using log-phase ribosomes, as shown in Fig. 3H. Does HPF contribute to this phenomenon? In a previous study, it was elucidated that HPF, which is expressed during the stationary phase and binds to 100S ribosomes exclusively, enhances the formation of 100S ribosomes by RMF (Ueta et al. 2005). On this basis, it is expected that HPF increases the dimer-formation activity of log-phase ribosomes by RMF. To elucidate the additional effects of HPF, HPF and RMF were simultaneously added to Log-HLR or Stat-HLR, as shown in Fig. 6. Although RMF hardly formed a dimer when using Log-HLR, as shown in Fig. 3C, a slight dimer was formed by the addition of HPF, as shown by the arrow in Fig. 6A. In comparison with Figs 3F and 6B, it can be seen that HPF stimulates 100S ribosome formation when using Stat-HLR. Despite plenty HPF, however, the amount of dimer in Log-HLR fell short of that in Stat-HLR when comparing Fig. 6A,B.


Figure 6
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  		Figure 6  Additional effects of HPF. RMF (RMF/ribosome = 10) and HPF (HPF/ribosome = 10) were simultaneously added to Log-HLR (A) and Stat-HLR (B). The mixtures were analyzed by SDGC. Sedimentation is from left to right. Arrow in (A) shows the position of the dimer. These results should be compared with Fig. 3C,F.

 
HPF does not interfere with IF3 activity, but stabilizes 100S ribosomes

HPF promotes the dimer formation activity of RMF. Does HPF interfere with the dissociation activity of IF3? To elucidate the relationship between IF3 and HPF, additional effects of these factors on Stat-HLR were examined. First, HPF (HPF/ribosome = 10) was added to Stat-HLR, as shown in Fig. 7A. As a result, HPF bound to 70S ribosomes, as shown by Western blotting, but the ribosome profile was unchanged in comparison with Figs 3D and 7A. This result shows that HPF does not have the ability to form dimers. IF3 (IF3/ribosome = 40) was then added, as shown in Fig. 7B. As a result, some 70S ribosomes dissociated to 30S and 50S subunits by the addition of IF3, in comparison with Fig. 7A,B. This ribosome profile was almost the same as when adding IF3 alone (Fig. 3E). Interestingly, HPF was removed from ribosomes by the addition of IF3, as shown by Western blotting. Next, IF3 was added, followed by HPF, as shown in Fig. 7C. This ribosome profile was the same as that in Fig. 3E, indicating that HPF does not interfere with the dissociation activity of IF3. Figure 7D shows the ribosome profile when RMF (RMF/ribosome = 10) and HPF (HPF/ribosome = 10) were added first, and then excess IF3 (IF3/ribosome = 80) was added. In comparison with Figs 6B and 7D, 100S and 70S ribosomes decreased and increased, respectively; however, significant amounts of 100S ribosomes remained in comparison with .Figs 5B and 7D. It is considered that the 100S ribosomes stabilized by HPF are hard to dissociate with IF3.


Figure 7
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  		Figure 7  HPF does not interfere with IF3 activity. Mixtures of factors and ribosomes were analyzed by SDGC. Sedimentation is from left to right. (A) HPF (HPF/ribosome = 10) was added to Stat-HLR. The binding of HPF to 70S ribosomes was examined by Western blotting as shown in the lower column. (B) HPF (HPF/ribosome = 10) was added, followed by IF3 (IF3/ribosome = 40). (C) IF3 (IF3/ribosome = 40) was added, followed by HPF (HPF/ribosome = 10). These results should be compared with Fig. 3D and 3E. (D) RMF (RMF/ribosome = 10) and HPF (HPF/ribosome = 10) were added, and then IF3 (IF3/ribosome = 80) was added. This result should be compared with Figs 5B and 6B.

 
Effect of polyamines on 100S ribosome formation

It is also known that ribosomal subunit dissociation and association are greatly affected by ionic conditions, such as magnesium ions, monovalent ions and polyamines (spermine, spermidine and putrescine) (Goss et al. 1982; Vila-Sanjurjo et al. 2004). Polyamines are present at millimolar concentrations in cells and are required for the efficient translation reaction. The concentration of polyamines is regulated by spermidine acetyltransferase, which increases in response to several stresses, such as high pH, cold shock and poor nutrient conditions. This means that polyamines decrease concomitantly with transition from the log to stationary phase. Moreover, it is reported that the accumulated spermidine due to the lack of spermidine acetyltransferase causes a decrease in 100S ribosomes during the stationary phase (Fukuchi et al. 1995; Raj et al. 2001). This suggests that polyamines relate in some way to ribosomal dimerization during transition from the log to stationary phase. To evaluate the effect of polyamines on ribosomal dimerization, RMF (RMF/ribosome = 10) and stationary-phase ribosomes were incubated for 30 min at 37 °C in polyamine buffer (5 mM Mg(Ac)2, 0.5 mM CaCl2, 5 mM NH4Cl, 95 mM KCl, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, pH7.3, 1 mM DTT). It is expected that stationary-phase ribosomes will change to log-phase ribosomes in polyamine buffer and ribosomal dimerization activity will decrease; however, 100S ribosomes could be formed in polyamine buffer as well as in association buffer (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the proteobacteria gamma group, including E. coli, most ribosomes are dimerized by RMF and HPF during the stationary phase (Wada et al. 1990). On the other hand, in the proposed ribosome cycle of bacteria, ribosomes which finished synthesizing proteins on mRNAs were dissociated to ribosomal subunits by ribosome recycling factor and EF-G in a GTP-dependent reaction, and then IF3 immediately bound to 30S subunits to stabilize the dissociation (Karimi et al. 1999; Hirokawa et al. 2005). According to this ribosome cycle, it seemed that ribosomal dimerization cannot occur during the stationary phase because RMF must bind to 70S ribosomes to form 100S ribosomes. What events occur before 100S ribosome formation? In order to elucidate ribosomal events before 100S ribosome formation, the relationships between protein factors (RMF and HPF) involved in 100S ribosome formation and IF3 involved in initiation complex formation were examined. First, the amount of IF3 in the cells was examined. As a result, the molar ratio of IF3 to ribosomes during the stationary phase was almost the same as that during the log phase, as shown in Fig. 1, indicating that 100S ribosomes can be formed by RMF and HPF in the presence of IF3 in the cell.

Next, to examine the dissociation activities of IF3 against log- and stationary-phase ribosomes, IF3 was added to log- or stationary-phase ribosomes in vitro. As a result, the dissociation activity of IF3 against stationary-phase ribosomes was lower than that against log-phase ribosomes, as shown in Fig. 3G. In contrast, the dimerization activity of RMF was higher against stationary-phase ribosomes than log-phase ribosomes, as shown in Fig. 3H, indicating that log-phase ribosomes become stationary-phase ribosomes, which are difficult to dissociate by IF3 and easy to convert into 100S ribosomes by RMF. Figure 4A shows that IF3 was difficult to bind to stationary-phase ribosomes. It is considered that this difference in the binding activity of IF3 between log- and stationary-phase ribosomes caused the lower dissociation activity of IF3 against stationary-phase ribosomes. On the other hand, Fig. 4B demonstrated no significant difference in the binding activity of RMF between log- and stationary-phase ribosomes. In a previous paper, it was predicted that the ribosomes would undergo large conformational changes when the 100S ribosome was formed (Yoshida et al. 2004); therefore, it is speculated that it is difficult for log-phase ribosomes to undergo conformational changes for 100S ribosome formation by RMF binding.

This indicates that ribosomes change for dimerization when they enter the stationary phase. What changes the ribosomes? It is possible that HPF changes the ribosomes, because HPF enhances 100S ribosome formation. To test this possibility, HPF was added to log-phase ribosomes with RMF in vitro, as shown in Fig. 6. As a result, HPF stimulated 100S ribosome formation using log-phase ribosomes as well as stationary-phase ribosomes; however, the amount of formed dimer fell short of that when using stationary-phase ribosomes. Also, HPF did not interfere with the dissociation activity of IF3 and was released from the ribosomes by IF3, as shown in Fig. 7. These results indicate that an unknown factor changes ribosomes for effective dimerization in addition to HPF.

We also examined the possibility that polyamines relate to ribosomal changes between log and stationary phases. RMF and stationary-phase ribosomes were incubated in polyamine buffer. From the results, it is considered that polyamines do not relate to the ribosomal changes dominating dimerization activity.

In conclusion, the experimental results in this study indicate that log-phase ribosomes become stationary-phase ribosomes, which are difficult to bind with IF3 and easily form 100S ribosomes by RMF and HPF; however, the causes are as yet unknown. It is reported that tmRNA (rescue-stalled ribosomes) and relA (synthesize ppGpp) interact with ribosomes in the transition period from the log to stationary phases (Wendrich et al. 2002; Ranquet et al. 2007). These interactions with ribosomes before dimerization might relate to ribosomal changes for effective dimerization. Future studies should aim to elucidate the nature and mechanism of ribosomal changes between log- and stationary phases by using the activities of IF3 and RMF as an indicator.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Quantitative analysis of IF3 by RFHR 2-D PAGE

Escherichia coli W3110 cells were grown in medium E (Vogel & Bonner 1956) containing 2% polypeptone and supplemented with 0.5% glucose at 37 °C with shaking at 100 cycles per minute. The cells in the log phase were harvested after 2.5 h of incubation. Stationary-phase-specific proteins derived from cells before inoculation did not remain in 2.5 h-cultured cells, and stationary-phase-specific proteins were still not expressed. Cells in the stationary phase were harvested after 4 days of incubation because the number of 100S ribosomes peaked at 4 days. Each cell pellet was ground with an approximately equal volume of quartz sand and then extracted with association buffer (100 mM CH3COONH4, 15 mM (CH3COO)2Mg•4H2O, 20 mM Tris–HCl at pH 7.6, and 6 mM 2-mercaptoethanol). A one-tenth volume of 1 M MgCl2 and two volumes of acetic acid were added to the cell extract, and the mixture was stirred for 1 h at 4 °C. After centrifugation at 5600 g for 10 min, the supernatant was dialyzed against 2% acetic acid three times. The proteins were lyophilized and stored at –80 °C until use. Two milligrams of lyophilized protein were analyzed by RFHR 2-D PAGE (Wada 1986). Protein spots on the gels were scanned with a GS-800 Calibrated Densitometer (Bio-Rad Laboratories Inc.). The optical density of the proteins was calculated as a function of their molecular weights, and the value of IF3 was normalized against those for ribosomal proteins near the IF3 spot on the gel.

Preparations of ribosomes

Ribosomes were prepared from E. coli W3110 cells. Crude ribosomes (CR) from the cells were prepared essentially as according to the method of Noll et al. (1973) with slight modifications, as described by Horie et al. (1981). CR was suspended in association buffer, which included several protein factors. Protein factors such as IF3 and RMF were removed from CR by ultracentrifugation through a sucrose cushion in high-salt buffer (1 M CH3COONH4, 15 mM (CH3COO)2Mg•4H2O, 20 mM Tris-HCl at pH 7.6, and 6 mM 2-mercaptoethanol). High-salt-washed ribosomes (HSR) include protein factors such as HPF. The protein factors were removed from the HSR by ultracentrifugation through a sucrose cushion in low-magnesium-ion buffer (100 mM CH3COONH4, 1 mM (CH3COO)2 Mg•4H2O, 20 mM Tris–HCl at pH 7.6, and 6 mM 2-mercaptoethanol). Finally, high-salt-washed and low-magnesium-treated ribosomes (HLR) were dialyzed against association buffer for use in the in vitro assay, and stored at –80 °C until use.

Preparations of IF3, RMF and HPF

The expression vector pQE-9 (Qiagen), which contains a 6 x His-tag at the N-terminus, was used to express His-tagged IF3 or His-tagged HPF. The ligated vectors were transformed to M15 strains. Expressions of His-tagged IF3 and HPF were induced by adding 0.3 mM and 0.1 mM IPTG (isopropyl-β-D(–)-thiogalactopyranoside), respectively. His-tagged IF3 and HPF were purified by a column filled with 1 mL nickel–nitrilotriacetic acid-agarose (Ni–NTA, Hi-Trap column from GE Healthcare) and then dialyzed against association buffer for use in the in vitro assay, and stored at –80 °C until use.

Native RMF was obtained from W3110{Delta}yfiA strains. More 100S ribosomes are formed in{Delta}yfiA cells than parental cells because YfiA inhibits the formation of 100S ribosomes (Ueta et al. 2005). The high-salt-washed supernatant fraction of 100S ribo-somes was separated by ultracentrifugation, which included native RMF. Native RMF was purified and concentrated using Centriprep YM-10K and YM-3K (Millipore) filters, dialyzed against association buffer for use in the in vitro assay, and stored at –80 °C until use.

Reactions of protein factors and ribosomes in vitro

The protein factors (IF3, RMF and HPF) were added to ribosomes (HLR) of 30 nmol prepared from cells in the log or stationary phase, and incubated for 30 min at 37 °C. When two factors were added to the HLR, the first factor was added and incubated for 30 min at 37 °C, and then the second factor was added and incubated for 30 min at 37°C. A sample solution of HLR without addition of the factor was also incubated as a control. The incubated samples were subjected to ultracentrifugation on 5–20% linear sucrose density gradients in association buffer. After centrifugation in a SW-41Ti rotor (Beckman) at 40 000 r.p.m. (about 20 000 g), for 90 min at 4 °C, ribosome profiles were observed at 260 nm by a UV-1700 spectrometer (Shimazu) using a flow cell. The experiments were performed more than three times for each condition.

Western blotting

After sucrose density gradient centrifugation, the samples were fractionated into 18 fractions. Proteins in each fraction were precipitated with 10% trichloroacetic acid (TCA), separated by 16% tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) (Schagger & von Jagow 1987), and transferred to PVDF membranes (Immobilon-FL transfer membrane, Millipore). IF3, RMF, and HPF were recognized by rabbit antisera against IF3, RMF, and HPF, respectively, which were detected with ECF substrate (GE Healthcare) using an FLA2000 imager (Fujifilm).


   Acknowledgements
 
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas ‘Spatio-temporal Network of RNA Information Flow’ (20 570 167) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

aPresent address: Department of Physics, Osaka Medical College, 2–7, Daigaku-Machi, Takatsuki, Osaka 569-8686, Japan Back

* Correspondence: yhide{at}art.osaka-med.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 13 March 2008
Accepted: 13 November 2008




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