Structure of the 100S Ribosome in the Hibernation Stage Revealed by ...

Structure

Article Structure of the 100S Ribosome in the Hibernation Stage Revealed by Electron Cryomicroscopy Takayuki Kato,1,2,5 Hideji Yoshida,3,5 Tomoko Miyata,1 Yasushi Maki,3 Akira Wada,4 and Keiichi Namba1,2,* 1Graduate

School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan Nanomachine Project, International Cooperative Research Project, Japan Science and Technology, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan 3Department of Physics, Osaka Medical College, 2-7 Takatsuki, Osaka 569-8686, Japan 4Yoshida Biological Laboratory, 31 Zushiokunawashiromoto, Yamashina, Kyoto 607-8081, Japan 5These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.str.2010.02.017 2Dynamic

SUMMARY

In the stationary growth phase of bacteria, protein biosynthesis on ribosomes is suppressed, and the ribosomes are preserved in the cell by the formation of the 100S ribosome. The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor and the hibernation promoting factor. However, the binding mode between the two 70S ribosomes and the mechanism of complex formation are still poorly understood. Here, we report the structure of the 100S ribosome by electron cryomicroscopy and single-particle image analysis. The 100S ribosome purified from the cell in the stationary growth phase is composed of two transfer RNA-free 70S ribosomes, has twofold symmetry, and is formed through interactions between their 30S subunits, where interactions between small subunit proteins, S2, S3 and S5, appear to be critical for the dimerization.

INTRODUCTION One of the most important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosome is a universally conserved, large ribonucleoprotein composed of two subunits. In bacteria, a small (30S) subunit and a large (50S) subunit associate to form a functional 70S ribosome; ‘‘S’’ is the unit of the sedimentation coefficient by analytical ultracentrifugation. The 30S subunit (0.9 MDa) is composed of one rRNA molecule called 16S RNA and ribosomal proteins S1–S21. The 50S subunit (1.8 MDa) is composed of two rRNA molecules, 5S and 23S, and ribosomal proteins L1–L36. Ribosomes can occupy as much as 45% of the total mass of bacterial cells, such as Escherichia coli, during their logarithmic (log) growth phase, in which the cells can actively synthesize various proteins. However, in the cells under stress conditions, such as starvation, ribosomal biosynthesis is

repressed, and protein synthesis is also suppressed. Such translational regulation mechanisms are very important for bacteria that have to survive harsh environments. In eukaryotes, it is known that the phosphorylation of initiation factor-2a (eIF2a) is a main adaptive mechanism for downregulating protein synthesis under stress conditions (Hinnebusch, 1994). In the proteobacteria gamma group, including E. coli, protein synthesis is mainly suppressed by the formation of 100S ribosomes (Wada et al., 1990). It has been found recently that the 100S ribosome is also formed in a gram-positive bacterium, Staphylococcus aureus (Ueta et al., 2010). The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor (RMF). The RMF is a basic, small protein (pI = 11.3; MW = 6507 Da), and its expression level remarkably increases during the transition from the log to the stationary phase. Another protein factor expressed during the stationary phase and binding to ribosomes is the hibernation promoting factor (HPF), which promotes the formation of the 100S ribosome by the RMF (Maki et al., 2000; Ueta et al., 2005). The 100S ribosome is formed during the stationary phase and has no translational activity (Wada et al., 1995). When the cells in the stationary phase are transferred to rich, nutritious culture media, the RMF is immediately released from the 100S ribosome, and the 100S ribosome dissociates back into 70S ribosomes (Wada, 1998). This process is rapid and is completed within 1 min (Aiso et al., 2005). After this process, the cells reinitiate protein synthesis and proliferation within 6 min. The E. coli mutant strain with rmf gene deletion cannot form 100S ribosomes, and the lifetime of this mutant is shorter than that of the wild-type (Yamagishi et al., 1993). These phenomena indicate that the interconversion between the 70S and 100S ribosomes is an important strategy for survival under stress conditions. The ribosomal resting stage, namely, the stage of forming 100S ribosomes, is an important part of the ribosome cycle and is called the hibernation stage (Yoshida et al., 2002). In previous studies, it has been reported that the RMF inactivates ribosomes by covering the peptidyl transferase center and the exit of the peptide exiting tunnel (Yoshida et al., 2002, 2004). It has also been suggested that the HPF binds to the 30S subunit at the subunit interface (Maki et al., 2000; Ueta et al.,

Structure 18, 719–724, June 9, 2010 ª2010 Elsevier Ltd All rights reserved 719

Structure Structure of the 100S Ribosome

Figure 1. Electron Cryomicrographs of Frozen-Hydrated 100S Ribosomes and Averaged Image (A) Noncrosslinked (left) and crosslinked (right) specimens. Red circles and yellow ellipses indicate monomer and dimer ribosomes, respectively. The scale bar represents 50 nm. (B) Average of 1156 particle images in one of the 40 different classes (Figure S1). The scale bar represents 20 nm.

2005). These results suggest that the RMF and HPF bind to the interface between the 50S and 30S subunits. However, electron microscopic observations of a negatively stained specimen suggested that the 100S ribosome is formed by the face-to-face contacts between the 30S subunits of the two 70S ribosomes, namely, as 50S-30S-30S-50S (Yoshida et al., 2002). The intercalation of the RMF and HPF in the subunit interface may induce conformational changes in the 30S subunits, causing two 70S ribosomes to dimerize. Moreover, previous studies suggested that the 70S ribosomes change their conformation before the dimerization during the transition from the log phase to the stationary phase so that the ribosomes in the stationary phase do not bind to initiation factor 3 (IF3) and easily form the 100S ribosome by binding the RMF and HPF (Yoshida et al., 2009). However, it is difficult to understand the formation mechanism of the 100S ribosome without its structural information. Here, we report the structure of the 100S ribosome in the hibernation stage obtained by electron cryomicroscopy cryoEM and single-particle image analysis. Our result revealed that the 100S ribosome is formed by two transfer RNA (tRNA)-free 70S ribosomes through the interactions between their 30S subunits, and that the S2, S3, and S5 proteins are likely to be directly involved in the interactions. RESULTS CryoEM Imaging, Classification, and Averaging We isolated 100S ribosomes from E. coli cells cultured for 4 days and collected cryoEM images in their frozen-hydrated state embedded in a thin vitreous ice film. Because the interaction between the 70S ribosomes in the dimer structure is very weak, the 100S ribosome easily dissociated into monomeric 70S ribosomes at the low concentration optimized for cryoEM observation to have appropriate particle densities in the images. Therefore, only a few 100S ribosome particles were found in each charge-coupled device (CCD) image, as indicated by yellow arrows in Figure 1A. We used a method called GraFix, which uses sucrose density gradient centrifugation with weak chemical fixation, to stabilize the 100S ribosomes for cryoEM observation and were able to obtain far more images of the 100S ribosome particle in each CCD image (Figure 1B). We picked up 11,887 particle images from 800 CCD images, classified them into 40 classes of different particle orientations by reference-free classification based on a singular value decomposition iterative classification scheme, and then aligned and averaged the particle images in each class (see Figure S1

available online). Typical averaged images clearly showed that the subunits in the 100S ribosome is aligned as 50S-30S-30S50S, as shown in Figure 1C. This result agrees with a previous EM observation of crosslinked 100S ribosomes negatively stained with uranyl acetate (Yoshida et al., 2002). It is now clear that the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by the face-to-face contacts between their 30S subunits. Structural Analysis of the 100S Ribosome We first tried to analyze the images of the entire 100S ribosome particle by a projection matching method, but the resolution of the obtained three-dimensional (3D) map was relatively low (30 A˚), and the map did not reproduce the structure of the 70S ribosome previously revealed by cryoEM image analysis (Gilbert et al., 2004) and X-ray crystallography (Zhang et al., 2009) (Figure S2), suggesting that the connection between the 30S subunits is relatively flexible. Therefore, we aligned the images of the 100S ribosome by using only one of the two 70S ribosome images. Then, the averaged image of the aligned 70S particle showed fine structural features, whereas the other 70S ribosome image was markedly blurred (Figure S3). The relative Euler angle and location between 70S ribosomes were widely distributed (Figure S4). These results strongly indicate the flexibility of the dimer connection of the 100S ribosome structure. Therefore, we treated the images of the two 70S ribosomes individually for 3D image reconstruction, but including the part corresponding to the 30S subunit of the other half to see the dimer connection in the final 3D image reconstruction. The resolution of the 3D map reconstructed from 17,461 images was 18 A˚ (Figure 2). Unfortunately, we were not able to identify the densities for the RMF and HPF in the 3D map, probably due to their small sizes and the limited resolution (Figure 2). Interestingly, however, it is clear that no obvious densities corresponding to the tRNA are present in the map (Figure 2B), suggesting that the 100S ribosome is composed of two tRNA-free 70S ribosomes. Model of the 100S Ribosome The obtained 3D density map clearly shows an extra mass (green) attached to the 30S subunit (yellow) of the 70S ribosome, as shown in Figure 2. The extra density corresponds to a part of the paired 70S ribosome. The map also confirmed the array of the 50S-30S-30S-50S subunits in the 100S ribosome particle (Figure 1C). Therefore, the extra density can be readily attributed to a part of the paired 30S subunit.

720 Structure 18, 719–724, June 9, 2010 ª2010 Elsevier Ltd All rights reserved


Figure 2. 3D Density Map of a 70S Ribosome with Part of Its Partner in the 100S Ribosome (A) The 50S and 30S subunits of a 70S ribosome and an extra density paired with the 30S subunit are colored cyan, yellow, and light green, respectively. (B) Cross-section showing the tRNA-free interface between the 50S and 30S subunits. The locations of the tRNA at the A-, P-, and E-site are indicated by the red, green, and yellow ribbon models, respectively. The scale bar represents 20 nm. Domains and proteins are labeled as: CP, central protuberance; L1, L1 protein of the 50S subunit; St, L7/L12 stalk; hd, head; bk, beak; sp, spur.

By comparing the features of the extra density with those of the 30S subunit, we were able to identify several characteristic density features shared by these two parts, such as the ‘‘beak’’ labeled bk in red in Figure 2. We built a density model of the 100S ribosome by fitting the 30S part of the 70S particle density into the extra density and making a pair of the 70S particle densities (Figure 3). We then determined the position and orientation of the two-fold symmetry axis from the coordinates of the two atomic models of the 70S ribosome docked into each of the pair as indicated in the right panel of Figure 3. The ‘‘head’’ regions are close to each other, whereas the ‘‘body’’ regions are relatively far apart. A density of the 30S subunit protruding toward the paired 30S subunit is connected to the part between the ‘‘head’’ and ‘‘body’’ of the pair as indicated by the dotted circle in Figure 3 and magnified in Figure 4. Thus, there are two points of contact between the 70S particles related by the two-fold symmetry axis in the 100S ribosome. Interactions between the Paired 30S Subunits To analyze the interactions between the paired 30S subunits in detail, we docked an atomic model of the 30S subunit (PDB code: 3I1M) into the corresponding regions of the 3D map of the 100S ribosome (Figure 3). The protruded density connected to the paired 30S subunit is attributed to ribosomal protein S2, which appears to be inserted into the pocket formed by three proteins, S3, S4, and S5 (Figure 4). The closest parts to this connection are two a helices of S3 (residues 107–126 and 128–142, colored red in Figure 4) and an a helix of S5 (residues 54–68, colored dark green in Figure 4) of the paired 30S subunit. The former helix of S3 mentioned above is not accommodated within the 3D map, and the density between S3 and S2 is not occupied by the model (indicated by arrow in Figure 4), suggesting that the binding of the RMF and HPF causes a conformational change of S3 for binding to S2 of the paired 30S subunit. In this model fitting, there is another density region that is not occupied by any component proteins of the ribosome near S6 and S18 (indicated by arrows in Figure 3). Because proteins S6 and S18 are important for the formation of a bridge between the 30S and 50S subunits as well as for the binding of IF3 (Gabashvili et al., 2000; Dallas and Noller, 2001), a structural change in this region may cause the inhibition of IF3 binding to the ribosome during the stationary phase (Yoshida et al.,

2009). Because the binding sites of RMF and HPF to the 70S ribosome have been identified to be at the interface of the 30S and 50S subunits (Maki et al., 2000; Yoshida et al., 2002, 2004), we cannot attribute this density to those protein factors. Although another protein called stationary-phase-induced ribosome-associated (SRA) protein has been found in the 100S ribosome by 2D-polyacrylamide gel electrophoresis (PAGE), this protein (MW = 5095 Da) may also be too small to be visualized at the present resolution (Izutsu et al., 2001). Therefore, the density might represent an unknown protein that cannot be detected by 2D-PAGE. However, because this extra density is the only change in the 30S structure observed in the hibernation state, it is still possible that part of this density represents the

Figure 3. Model of the 100S Ribosome with a Two-Fold Symmetry The 50S subunits are colored cyan and pink, and the 30S subunits are colored yellow and green. The two-fold symmetry axis is indicated in pink in the right panel. The arrows indicate the extra density unoccupied by any known component proteins of the ribosome. The black and red dots in the right panel indicate the position of S6 and S18, respectively. Domain labels not shown in Figure 2 are: bd, body; pt, platform.



Figure 4. Contact Point between the 70S Ribosomes (A) Paired 30S subunits are colored yellow and green. (B) The slab of the part indicated by the box in (A) is viewed in stereo, in the same direction as in (A) in the upper panel and in the direction of the arrow in (A) in the lower panel, to show the S2-S5 contact region more clearly. The S2, S3, S4, and S5 proteins are shown by the orange, pink, purple, and cyan ribbon models, respectively. Two a helices of S3 (residues 107–126 and 128–142) and one a helix of S5 (residues 54–68) are colored red and dark green, respectively. Domain labels are the same as in Figures 2 and 3.

factors that promote the formation of the 100S ribosome, such as RMF and HPF. DISCUSSION The translation process in bacteria can be divided into four stages: initiation, elongation, termination, and recycling (Schmeing and Ramakrishnan, 2009). The transition from the 70S ribosome in the log phase to the100S ribosome in the stationary phase is believed to occur between the recycling stage and the next initiation stage (Yoshida et al., 2009). The process between these two stages has been proposed as follows. After releasing the nascent polypeptide from the ribosome by release factors in the termination stage, the posttermination complex is formed by three components: mRNA, tRNA, at the P/E-site, and the 70S ribosome. In the first step of the recycling process, the posttermination complex disassembles into the 50S subunit and the mRNA-tRNA-30S complex by the ribosome recycling factor (RRF) and the elongation factor G (EF-G). In the second step, the mRNA and the tRNA are released from the complex by the binding of IF3 to the complex. Then, protein biosynthesis is restarted by the binding of the initiator fMet-tRNAfMet, mRNA, IF1, and IF2 to the 30S subunit in the initiation stage (Schmeing and Ramakrishnan, 2009). Because our present study showed that the 100S ribosome does not contain any tRNAs (Figure 2B), the 100S ribosome must be formed by ribosomes that have released the tRNA by IF3 in the above canonical ribosome cycle. However, it has been reported that the binding of IF3 to the ribosome inhibits the formation of the 100S ribosome (Yoshida et al., 2009). These results indicate that the stage of 100S ribosome formation (the hibernation stage) cannot be described in the canonical ribosome cycle. Our present study suggests that the scheme of ribosome cycle should be modified as shown in Figure 5. The binding of the RMF and HPF to the ribosome for the formation of the 100S ribosome must occur before the binding of IF3 to the ribosome. It is speculated that the transition from the 70S ribosome to the 100S ribosome starts before or after the recycling stage by

RRF and EF-G. In both cases, tRNA and mRNA must be released by some factors other than IF3, which may be RMF and/or HPF, or other protein factors. In the 100S ribosome, the protruding density identified as S2 of the 30S subunit is close to the entrance for mRNA formed by S3, S4, and S5 in the paired 30S subunit. The formation of the 100S ribosome appears to induce the conformational change of S3 and/or S5 for binding to S2 in the paired 30S subunit. A recent structural study suggested that S2 is stabilized by the Shine-Dalgarno helix docked in the chamber between the ‘‘head’’ and the ‘‘platform’’ of the 30S subunit (Kaminishi et al., 2007). However, mutation studies revealed that S3 and S4 have helicase activity for mRNA (Takyar et al., 2005), and that S4 and S5 play an important role for the translational fidelity (Ito and Whittmann, 1973; Piepersberg et al., 1975). The functions of proteins S2, S3, S4, and S5 are related to the binding of mRNA to the 30S subunit and the movement of mRNA in the 70S ribosome. Even during the stationary phase a modest level of gene expression is present, suggesting that a relatively small amount of translatable mRNA and the active 70S ribosome coexist with a large amount of the 100S ribosome in the cell during the stationary phase. If mRNA could bind to the 100S ribosome, the modest level of gene expression would be inhibited. In order to control the translational activity in the stationary phase, undesirable binding of mRNA to the ribosomes may be inhibited by the conformational changes around the S2-S5 region upon formation of the 100S ribosome. If the extra density that appears in the 30S subunit of the 100S ribosome (Figure 3) represents the protein factors that promote the hibernation state, its relatively close location to the S2-S5 regions may account for the possible conformational change in that region for 100S ribosome formation. A higher-resolution structure is, however, necessary to confirm this possibility. Our present study also revealed that the interactions between the 30S subunits in the 100S ribosome are very weak and loose. Because the transition from the 100S ribosome to the 70S ribosome must occur immediately when the cells are released from environmental stresses, such as starvation, we suggest that the weak and loose binding between the 30S subunits is essential for this quick recovery of protein translation after surviving harsh environments.



Figure 5. Schematic Diagram Showing the Formation Process of the 100S Ribosome upon Transition from the Log Phase to the Stationary Growth Phase Protein factors involved in the process are: RRF, ribosome recycling factor; EF-G, elongation factor G; RMF, ribosome modulation factor; HPF, hibernation promoting factor.

TemCam-F415MP slow-scan CCD camera (image pixel size: 1.7 A˚/pixels) and at 1.3–3.5 mm underfocus. The electron dose was set to 20 e /A˚2.

EXPERIMENTAL PROCEDURES 100S Ribosome Preparation Escherichia coli W3110 cells were grown in medium E (Vogel and Bonner, 1956), which contained 2% polypeptone and was supplemented with 0.5% glucose. Cells were shaken at 100 cycles per minute at 37 C. Cells in the stationary phase were harvested after 4 days of incubation because the level of the 100S ribosome peaked at 4 days when exposed the above-mentioned growth conditions. Crude ribosomes from the cells were prepared essentially as according to the method of Horie et al. (1981) and were suspended in a buffer called the association buffer (77 mM KCl, 15 mM (CH3COO)2Mg, 20 mM HEPES-KOH [pH 7.6]). The 100S ribosomes were mildly fixed by the GraFix method with slight modifications to suppress their dissociation into 70S ribosomes at a low protein concentration optimized for electron cryomicroscopic observation (see Figure S1) (Kastner et al., 2008). The low (5%) and high (20%) sucrose density solutions in the association buffer were prepared, and then 0.25% glutaraldehyde (EM grade 25%, Nacalai tesque, Japan) was added only to the high sucrose density solution. Sucrose gradients were prepared by using a gradient maker (Gradient mate, Towa Labo, Japan). At first, a 3% sucrose density solution in the association buffer was applied on top of the gradient to avoid an undesirable fixation reaction, and then the solution of 60 nmol crude ribosomes was applied on top of it. After centrifugation with a SW-41Ti rotor (Beckman) at 40,000 rpm (about 20,000 3 g) for 90 min at 4 C, the band profile of the ribosome was observed at 260 nm by a UV-1700 spectrometer (Shimazu) with a flow cell. Under the above-described conditions, nonspecific, artificial dimers of 70S ribosomes were not formed, as shown in Figure S2B. After the sucrose density gradient centrifugation for fixation, the fractions containing 100S ribosomes were collected and diluted with the association buffer. The solution was again subjected to ultracentrifugation on a 5% 20% linear sucrose density gradient in the association buffer without the fixation reagent. The crosslinked 100S ribosomes were purified in this step because unfixed ones were dissociated into 70S ribosomes under the ribosomal concentration of about 4 nmol, as shown in Figure S1. Finally, the sucrose in the solution was removed by using a centrifugal filter (Amicon Ultra 100K, Millipore).

Electron Cryomicroscopy A 3 ml sample solution of the crosslinked 100S ribosome in a buffer containing 20 mM HEPES-KOH (pH 7.6), 15 mM (CH3COO)2Mg, 77 mM KCl was applied onto a holey carbon film on a molybdenum grid (Quantifoil R0.6/1.0). After excess solution was blotted by the filter papers from both sides, the grid was quickly frozen by rapidly plunging it into liquid ethane by using FEI Vitrobot. The specimen was observed at a temperature from 50 K to 65 K by using a JEOL JEM-3200FSC electron cryomicroscope equipped with a liquid helium-cooled spacemen stage, a U-type energy filter, and a field-emission electron gun, which was operated at 200 kV. Zero-loss images were recorded under low-dose conditions at a magnification of 88,0003 by using a TVIPS

Image Processing The defocus and astigmatism in each image was determined by using CTFFIND3 (Mindell and Grigorieff, 2003). In total, 11,887 particle images of the 100S ribosome with a clear feature of the dimer structure were manually collected by using BOXER in the EMAN package. The phase-flipped images were used in the following image processing (Ludtke et al., 1999). The images of the 100S ribosome were classified into 40 different classes, and then they were aligned and averaged in each class by using refine2d.py in EMAN, which is a program for classification based on the singular value decomposition iterative classification scheme. Additional image processing was carried out by using the SPIDER software package (Frank et al., 1996) on a PC cluster with 60 CPUs (RC server Calm2000, Real computing, Tokyo, Japan). In order to avoid misalignment of the ribosome images by a possible flexibility of the dimer, we carried out single-particle image analysis on each 70S ribosome particle of the dimer. First, the individual particle images of the 100S ribosome were cropped in a box of 360 3 360 pixels, and then each of the two 70S ribosome particle images with part of the dimer pair was extracted in a box of 256 3 256 pixels with circular mask, then centered. The initial number of such particle images used for the following image analysis was 23,774, and the final 3D map was reconstructed from 17,461 particle images. The resolution was estimated to be 18 A˚ by the Fourier shell correlation with a 0.5 threshold. The amplitude of the structure factor was corrected by using X-ray solution scattering intensity data (Gabashvili et al., 2000) for the final reconstruction. The visualization of the 3D map and the fitting of the atomic model were carried out by UCSF Chimera (Pettersen et al., 2004). ACCESSION NUMBERS The 3D density map of the 100S ribosome has been deposited in the Electron Microscopy Data Bank with accession code EMDB-5174. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and can be found with this article online at doi:10.1016/j.str.2010.02.017. ACKNOWLEDGMENTS We thank Izumi Tokita, Masaaki Urabe, and JEOL Ltd. for maintaining the electron cryomicroscope at the best possible condition. T.K. is funded by the Global Center of Excellence Program to the Graduate School of Frontier Biosciences, Osaka University. This work was supported in part by Grantsin-Aid for Scientific Research to K.N. (16087207 and 21227006), and H.Y. (20570167) and T.M. (30423156) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received: January 24, 2010 Revised: February 19, 2010 Accepted: February 26, 2010 Published: June 8, 2010



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