- Email: [email protected]
Movement of the Decoding Region of the 16 S Ribosomal RNA Accompanies tRNA Translocation
doi:10.1006/jmbi.2000.4213 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 304, 507±515
COMMUNICATION
Movement of the Decoding Region of the 16 S Ribosomal RNA Accompanies tRNA Translocation Margaret S. VanLoock1, Rajendra K. Agrawal2,3, Irene S. Gabashvili2 Li Qi2,3, Joachim Frank3,4 and Stephen C. Harvey1* 1
Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham, Birmingham AL 35294-0005, USA 2
Wadsworth Center
3
Department of Biomedical Sciences, State University of New York at Albany 4
Howard Hughes Medical Institute, Health Research Inc. at the Wadsworth Center Empire State Plaza, PO Box 509, Albany, NY 12201-0509, USA *Corresponding author
The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)2 complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately Ê toward the P-site. This implies that elongation factor G actively 8A pushes both the decoding site and the mRNA/tRNA complex during translocation. # 2000 Academic Press
Keywords: ribosome; elongation factor; decoding site; translation; translocation
The ribosome is the site of protein synthesis in the cell. In bacteria, it is composed of two unequal subunits: the smaller 30 S subunit, which is responsible for decoding the messenger RNA (mRNA), and the larger 50 S subunit, which catalyzes peptide bond formation. The ribosome has three principal transfer RNA (tRNA) binding sites between the subunits (Grajevskaja et al., 1982; Kirillov et al., 1983; Lill et al., 1984; Rheinberger et al., 1981): the aminoacyl site (A-site), the peptidyl site (P-site), and the exit site (E-site). Following peptide bond formation (transpeptidation), hydrolysis of GTP by elongation factor G (EF-G) is used to drive the translocation of the peptidyl tRNA from the A-site to the P-site and, simultaneously, the transport of the uncharged tRNA from the P-site to the E-site. However, translocation at substantially reduced levels can occur in the absence of EF-G (Spirin, 1985). Abbreviations used: EF-G, elongation factor G, cryoEM, cryo-electron microscopy. E-mail address of the corresponding author: [email protected] 0022-2836/00/040507±9 $35.00/0
Previous structural work on individual ribosomal proteins and some fragments of ribosomal RNAs (Agrawal & Frank, 1999; Moore, 1998; Ramakrishnan & White, 1998; Woodson & Leontis, 1998), coupled with the recent progress on crystal structure determination of the ribosomal 30 S subunit (Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000), the 50 S subunit (Ban et al., 1998, 1999, 2000), and the intact 70 S ribosome (Cate et al., 1999) from the organisms living in extreme environments (i.e. halophilic archeon and thermophilic bacteria) lead to optimism that the atomic details of some key interactions will soon be revealed. This information is supplemented by cryo-electron microscopy (cryo-EM), which has yielded lower-resolution information on various ribosomal complexes in different functional states during the translational elongation cycle, revealing important conformational changes (Agrawal et al., 1996, 1998, 1999a,b, 2000; Frank & Agrawal, 2000; Gabashvili et al., 2000; Stark et al., 2000), and providing clues to the underlying interatomic interactions. One of the major goals of ribosome research is the elucidation of the structural basis for the mechanism of EF-G-dependent tRNA trans# 2000 Academic Press
508
Movement of the Decoding Region Accompanies Translation
Movement of the Decoding Region Accompanies Translation
location. Here, we show that data from a variety of high-resolution and low-resolution experiments can be integrated into models that address this issue, using a combination of manual and automated molecular modeling methods. In the 30 S ribosomal subunit, the anticodon ends of the A and P-site tRNAs bind in the highly conserved 1400-1500 region of helix 44 in the 16 S rRNA, referred to as the decoding-site region (inset in Figure 1(a)). The decoding site, together with helix 27, the 530 loop, and 1050 region of the 16 S rRNA, allows the ribosome to distinguish between cognate and non-cognate tRNAs in the A-site. However, given that the density for helix 44 is well de®ned in a variety cryo-EM maps (Agrawal et al., 1996, 1998, 1999a,b, 2000; Frank & Agrawal, 2000; Gabashvili et al., 2000; Stark et al., 2000) and that there is an atomic structure for the A-site region of this helix (Fourmy et al., 1996, 1998), we have focused our efforts on understanding the role of helix 44 and the A-site region of the decoding site in both ribosomal ®delity (VanLoock et al., 1999) and tRNA translocation. The contributions of other regions of the 16 S rRNA in these processes will begin to be deciphered as more detailed structural information becomes available. We developed an atomic model for helix 44 of the Escherichia coli 16 S rRNA (Figure 1(b)) using the phosphate positions for this helix from the Thermus thermophilus ribosomal crystal structures (Cate et al., 1999; Clemons et al., 1999) as a guide. Adjustments were made as needed so that the helix ®ts properly into Ê resolution cryo-EM map of the E. coli the 11.5 A 70 S ribosomal initiation complex (Gabashvili et al., at the riboso2000), which contains fMet-tRNAMet f mal P-site, and to accommodate sequence and secondary structure differences in this region between E. coli and T. thermophilus (Gutell, 1994). In the modeling we paid particular attention to the structure of the critical A-site region at the top of helix 44 (Figure 1(c)), which has been studied extensively. An RNA oligomer with a sequence identical to the E. coli A-site region shows tRNAdependent footprinting patterns very similar to those for the A-site in the intact ribosome (Purohit & Stern, 1994), and the structure of a similar oligo-
509 mer alone and complexed with the antibiotic paromomycin has been determined by NMR (Fourmy et al., 1996, 1998). The backbone conformation of the A-site region has been determined crystallographically, for both the isolated 30 S subunit (Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000) and for the intact 70 S ribosome with bound tRNA (Cate et al., 1999). Phosphate positions in both NMR structures (Fourmy et al., 1996, 1998) and the crystal structures (Cate et al., 1999; Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000) are all very similar. In addition, a previously published model of the A-site region of the 16 S rRNA complexed with A and P-site tRNAs and mRNA (VanLoock et al., 1999) agrees very well with the experimentally determined structures, with a rootÊ for mean-square deviation (RMSD) of less than 1 A the phosphate positions. Interestingly, the calcuÊ resolution electron density map of the lated 7.8 A previous decoding site model (VanLoock et al., 1999) is strikingly similar to that reported for the Ê resolution 70 S ribosomal crystal structure 7.8 A (Cate et al., 1999), with a substantial hole (see Figure 6 by Cate et al., 1999) around nucleotides A1492 and A1493 in both electron density maps. Here, we used an NMR structure for an A-site RNA oligomer in the absence of aminoglycoside (Fourmy et al., 1998) to analyze conformational changes in the decoding site upon tRNA translocation. Ê We docked the model for helix 44 into the 17 A resolution cryo-EM map of the 70 S ribosome in a pre-translocation complex containing both A and P-site tRNAs and mRNA. This new three-dimensional reconstruction was obtained from a complex at the that was prepared by binding tRNAMet f P-site and Phe-tRNAPhe at the A-site of an MF-mRNA programmed ribosome (Gabashvili et al., 2000) (R.K.A, L.Q & J.F., unpublished results). No adjustments to the position of helix 44 were necessary, suggesting that its conformation is very similar to that in the initiation complex. In the pre-translocational 70 S (tRNA)2 complex, the density for the A and P-site tRNAs is very clear (Figure 2(a) and (b)). We docked the X-ray structure (Sussman et al., 1978) of tRNAPhe into the P-site tRNA density and a model for the
Figure 1. The structure of the decoding site region of the ribosome. (a) Secondary structure diagram of the E. coli 16 S rRNA (Gutell, 1994) with helix 44 highlighted in cyan, and the sequence of the decoding site region enlarged in Ê resolution, of the 30 S ribosomal subunit the inset. (b) Ribbons image (Carson, 1987) of the cryo-EM map, at 11.5 A isolated from the E. coli 70 S ribosomal initiation complex (Gabashvili et al., 2000). Helix 44 is highlighted in cyan and the placement of the atomic model for the entire helix 44 is shown in purple. The black box indicates the decoding-site region of helix 44. The EM map is shown at higher threshold values to emphasize the shape and location of the helix 44. At lower threshold values, the entire structure of helix 44 is surrounded by density. (c) The region has been enlarged and displayed in stereo to show the structural details. The structure displayed in the electron density corresponding to the A-site was taken from an NMR structure of an RNA oligomer mimicking the A-site region of the decoding site (Fourmy et al., 1998). The phosphate positions in this structure are very similar to that of both a model for the decoding site in a complex with A and P-site tRNAs and mRNA and the X-ray crystal structures of the isolated 30 S subunit (Clemons et al., 1999) and the intact 70 S ribosome with bound tRNA (Cate et al., 1999). The enlarged area is the primary focus of this article, and the remaining Figures involving the A-site structure are shown from this view.
510
Movement of the Decoding Region Accompanies Translation
Figure 2. Stereo view showing the ®t of an NMR structure of an A-site RNA oligomer (Fourmy et al., 1998) in the cryo-EM density. (a) Fitting of the A-site region of the decoding site (blue) into the cryo-EM density of the pre-translocation 70 S(tRNA)2 complex (grey). Density corresponding to the P-site tRNA (green) is also visible in this ribosomal reconstruction. The EM density is shown at higher threshold values for clarity. At lower threshold levels, the atomic structure of the tRNAs is fully covered by EM density. (b) Same view as in (a), with a codon of mRNA (cyan) added, but without the EM density map displayed. The A-site mRNA-tRNA complex lies on the major-groove side of the decodingsite helix in close proximity to the highly conserved nucleotides A1492 and A1493. These nucleotides are thought to be important for mRNA proofreading and ribosomal ®delity (Fourmy et al., 1996; VanLoock et al., 1999). Landmarks: A, A-site tRNA; P, P-site tRNA.
A-site tRNA complexed with a three-nucleotide poly(U)mRNA (Easterwood et al., 1994) into the density corresponding to the A-site tRNA. The orientation of the A and P-site tRNAs is very simi-
lar to that reported on in the X-ray crystal structure of the 70 S ribosome (Cate et al., 1999), with only slight variations in the A-site tRNA position. In this complex, the A-site codon-anticodon complex
Movement of the Decoding Region Accompanies Translation
lies on the major groove side of the decoding site helix, in close proximity to the universally conserved nucleotides A1492 and A1493 (Figure 2(b)). This is consistent with previous studies suggesting that these nucleotides may interact directly with the mRNA in the decoding process, and that this interaction may contribute to translational ®delity (Fourmy et al., 1996; VanLoock et al., 1999). The importance of these nucleotides is indicated by the ®nding that mutation or deletion of A1492 and A1493 is lethal to the cell (Yoshizawa et al., 1999). We analyzed the A-site region of the decoding site from cryo-EM density maps of the 70 S ribosome in the pre-translocational state with EF-G and a non-hydrolyzable GTP analog with bound A and P-site tRNAs (Agrawal et al., 1999a), the 70 S (tRNA)2 EF-G GMPPCP complex, and in a complex with EF-G GDP and fusidic acid with bound P and E-site tRNAs (Agrawal et al., 1999a), the 70 S (tRNA)2 EF-G GDP fusidic acid complex. These cryo-EM maps provide a snapshot of the ribosome immediately after peptide bond formation (pre-translocation complex), upon binding of EFG-GTP to the ribosome (70 S (tRNA)2 EFG GMPPCP complex), and just after tRNA translocation, but prior to EF-G dissociation from the ribosome (70S (tRNA)2 EF-G GDP fusidic acid complex). We docked the model for helix 44 into the cryo-EM maps. The lower two-thirds of helix 44 ®ts very nicely into all of these maps. The A-site region of helix 44 ®ts well into the density for the 70 S (tRNA)2 EF-G GMPPCP complex but poorly into the density for the 70 S (tRNA)2 EFG GDP fusidic acid complex. We made the appropriate changes necessary to ®t the model into the electron density map for the latter state, to understand better the nature of the conformational changes associated with tRNA translocation. The density corresponding to the A-site region of the decoding site moves toward the P-site region Ê when comparing the by approximately 8 A 70 S EF-G GDP fusidic acid complex to the pretranslocation and 70 S EF-G GMPPCP complexes (Figure 3(a)). In order to accommodate this change, the turn in the upper part of helix 44 around nucleotides 1410-1490 rotates slightly, and the entire decoding-site structure shifts toward Ê the P-site tRNA-binding site by approximately 8 A (Figure 3(b) and (c)). As a result of the conformational change, nucleotides A1492 and A1493 Ê toward the P-site move by approximately 12 A tRNA-binding region (Figure 3(c)). This shift corresponds roughly to three nucleotides or one codon. Similar shifts in the P-site region of the decoding site density are also visible, but since its structure is not well de®ned, the exact nature of the shifts is unclear. Previous studies have suggested that the mass center of the A and P-site tRNAs moves Ê during translocation by approximately 12 A (Wadzack et al., 1997), and that the mRNA is shifted by exactly one codon (Beyer et al., 1994). The current results thus suggest that during translocation, the decoding site region moves in the
511 same direction and by approximately the same amount as the A and P-site tRNAs. In order to understand the role of EF-G in translocation, we displayed the previously (Agrawal et al., 1999a) ®tted X-ray structure of EF-G (Czworkowski et al., 1994) in the cryo-EM map of the 70 S EF-G GDP fusidic acid complex (Figure 3(b)). In this complex, domain IV of EF-G binds in the region that was occupied by the A-site tRNA anticodon stem loop in the pre-translocation complex (compare Figure 2(a) with 3(b): see also Agrawal et al., 1999a). In addition, the tip of EF-G domain IV lies in a patch of the ribosomal molecular envelope previously occupied by the A-site region of helix 44 in the pre-translocation complex (Figures 2(a) and 3(b)). It is known that amino acid mutations or deletions in the tip of domain IV signi®cantly decrease the rate of tRNA translocation (Savelsbergh et al., 2000), suggesting that it is critical for EF-G function. The location of EF-G domain IV in the 70 S EF-GGDP fusidic acid complex is highly suggestive of its function. During translocation, domain IV may actively push both the decoding site and the mRNA-tRNA complex from the A-site to the P-site. The tip of EF-G domain IV may also serve as a placeholder by occupying the A site and preventing slippage of the mRNAtRNA complex back into the A-site before translocation is complete. Both of these mechanisms would be consistent with the ®nding that deletion of EF-G domain IV reduces the rate of translocation 1000-fold, without affecting EF-G binding or GTP hydrolysis (Rodnina et al., 1997). Once EF-G dissociates from the ribosome, the decoding site is free to move back to its original position and accept a new incoming A-site tRNA. The back-and-forth movement of the decoding site region during translocation is consistent with suggestions that the decoding site acts as a molecular ratchet that carries the mRNA through the 30 S mRNA channel (VanLoock et al., 2000). Indeed, recent cryo-EM studies have shown that the entrance to the mRNA channel in the 30 S subunit opens following EF-G GTP binding, and that, during translocation, the 30 S subunit undergoes a ratchet-like rotation that follows the direction of tRNA movement relative to the 50 S subunit (Frank & Agrawal, 2000). Together, these data suggest that the ribosome actively participates in the translocation of the mRNA-tRNA complex from one tRNA binding site to another, and that the observed conformational changes seen in the tRNA-binding regions of the 30 S subunit consistently follow the direction of tRNA movement in the ribosome. Ê shift of the decoding region of helix 44 The 8 A during translocation may be necessary to help lower the energy barrier associated with tRNA translocation. The energy freed up by EF-G-dependent GTP hydrolysis is used to induce a conformational change in the A-site that may serve to position the A-site tRNA such that it can ef®ciently move into the P-site and, therefore, lower the
512
Movement of the Decoding Region Accompanies Translation
Figure 3 (legend opposite)
Movement of the Decoding Region Accompanies Translation
513
Figure 4. Stereo view of superimposed models of helix 44 in the pre and post-translocation complexes. The pretranslocation 70 S(tRNA)2 complex is shown in blue and the 70 S (tRNA)2 EF-G GDP fusidic acid complex in red (Agrawal et al., 1999a). The lower portion of helix 44 remains ®xed while its tip, the decoding region, undergoes signi®cant conformational changes upon EF-G-dependent GTP hydrolysis and tRNA translocation. The arrow indicates the direction of the movement of the A (pink) and P-site (green) tRNAs, the mRNA (cyan), and the decoding region during tRNA translocation.
Figure 3. Stereo view of the cryo-EM densities from the decoding site region of the pre and post-translocation complexes. (a) Comparison of the density for the A-site region of helix 44 from cryo-EM maps of the pre-translocation 70 S (tRNA)2 complex (black) and the 70 S (tRNA)2 EF-G GDP fusidic acid complex (Agrawal et al., 1999a) (cyan). There is a signi®cant shift in the electron density for the A-site region of helix 44 toward the P-site tRNA-binding domain upon EF-G-dependent GTP hydrolysis. (b) Fitting of the structure for the A-site region of the 16 S rRNA decoding site (Fourmy et al., 1998) (red) into the cryo-EM map (cyan) of the 70 S (tRNA)2 EF-G GDP-fusidic acid complex. The structure of the decoding site as shown in Figure 2 was altered from that in the pre-translocation comÊ towards the P-site. A major portion of domain IV of EF-G (yellow) plex and was shifted by approximately 8 A occupies the same position occupied previously by the A-site tRNA in the pre-translocation complex (see Agrawal et al., 1999a) (Figure 2(a)), and the tip of domain IV lies in the electron density previously occupied by the 16 S rRNA decoding-site region in both the pre-translocational and the 70 S (tRNA)2 EF-G GMPPCP complexes. In this reconstruction, the A-site tRNA (pink) seen in the pre-translocation complex (Figure 2) has been moved to the P-site upon EF-G-dependent GTP hydrolysis. (c) Comparison of the decoding-site structures from the pre-translocation complex (blue) and the 70 S (tRNA)2 EF-G GDP fusidic acid complex (red). In this transition, the A-site region shifts about Ê toward the P-site tRNA-binding domain, and rotates slightly. As a consequence of these combined motions, 8A Ê toward the P-site, roughly the length of one codon. A1492 and A1493 are moved approximately 12 A
514 activation energy barrier of tRNA translocation. In the absence of EF-G the decoding site may fail to undergo the observed conformational changes. However, some small fraction of tRNAs may still overcome the energy barrier associated with translocation and actively translocate on their own, a phenomenon that has been observed experimentally (Spirin, 1985). The observed movement in the A-site region of the decoding site is in line with previous suggestions that the ribosome has a moveable domain (Dabrowski et al., 1998) that drives translocation of tRNA. The lower portion of helix 44, which remains motionless throughout translocation (Figure 4), may serve as an anchoring point for the decoding site region. During translocation, EF-G binds to the ribosome and its GTP-binding domain interacts with the a-sarcin-ricin loop on the 50 S subunit (Agrawal et al., 1999a). Upon GTP hydrolysis, the a-sarcin-ricin loop may undergo a conformational change that alters its interactions with the large subunit protein L14 which forms an intersubunit contact with helix 44 of the 30 S subunit (Gabashvili et al., 2000). These conformational changes may in turn affect the conformation of the decoding site. A second contact between the decoding site and the 1920 region of the 50 S subunit, referred to as bridge B2a (Gabashvili et al., 2000), might also be involved in the binding of EF-G and tRNA translocation. The density around this contact changes upon EF-G-dependent GTP hydrolysis, and nucleotides in this region of the 50 S are protected from chemical attack in the presence of EF-G GDP and fusidic acid (Wilson & Noller, 1998). Conformational changes in the A-site during translocation would also help explain the decreased tRNA translocation rate in the presence of aminoglycoside antibiotics (Davies & Davis, 1968). Binding of aminoglycosides to the decoding site may lock the A-site in the conformation observed before EF-Gdependent GTP hydrolysis, therefore preventing structural changes in this region which are necessary for ef®cient tRNA translocation. Taken together, these results strongly suggest that, after transpeptidation, the energy from EF-Gdependent GTP hydrolysis is used by the ribosome to actively push both the decoding site and the mRNA-tRNA complex during translocation. This movement is consistent with the proposal that the ribosome is acting as a molecular ratchet (Frank & Agrawal, 2000; VanLoock et al., 2000). Materials and methods Atomic models for helix 44 were developed manually using a combination of molecular modeling packages including Insight II, Quanta (both products of Molecular Simulations Inc.), and O (Jones et al., 1991). Coordinates for the models will be made available from http://uracil.cmc.uab.edu/Publications. Coordinates for the P-site tRNA were taken from the X-ray structure of tRNAPhe (Sussman et al., 1978). The
Movement of the Decoding Region Accompanies Translation tRNAs were docked into their corresponding binding sites in each of the ribosomal cryo-EM reconstructions. Coordinates for the A-site tRNA and mRNA were taken from a previously published model of the A-site tRNA/ mRNA complex (Easterwood et al., 1994).
Acknowledgments The authors thank Scott Stagg, Christian Spahn and Pawel Penczek for helpful discussions. The work was supported by grants from the National institutes of Health: R01 GM53827 (S.C.H.), R37 GM29169 (J.F.), and R01 GM 55440 (J.F.).
References Agrawal, R. K. & Frank, J. (1999). Structural studies of the translational apparatus. Curr. Opin. Struct. Biol. 9, 215-221. Agrawal, R. K., Penczek, P., Grassucci, R. A., Li, Y., Leith, A., Nierhaus, K. H. & Frank, J. (1996). Direct visualization of A, P, and E-site transfer RNAs in the Escherichia coli ribosome. Science, 271, 1000-1002. Agrawal, R. K., Penczek, P., Grassucci, R. A. & Frank, J. (1998). Visualization of elongation factor G on the Escherichia coli 70 S ribosome: the mechanism of translocation. Proc. Natl Acad. Sci. USA, 95, 61346138. Agrawal, R. K., Heagle, A. B., Penczek, P., Grassucci, R. A. & Frank, J. (1999a). EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70 S ribosome. Nature Struct. Biol. 6, 643-647. Agrawal, R. K., Lata, R. K. & Frank, J. (1999b). Conformational variability in Escherichia coli 70 S ribosome as revealed by 3D cryo-electron microscopy. Int. J. Biochem. Cell Biol. 31, 243-254. Agrawal, R. K., Heagle, A. B. & Frank, J. (2000). Studies of elongation factor G-dependent tRNA translocation by three-dimensional cryo-electron microscopy. In The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions (Garrett, R. A., Douthwaite, S. R., Liljas, A., Matheson, A. T. & Moore, P. B., eds), pp. 53-62, ASM Press, Washington, DC. Ban, N., Freeborn, B., Nissen, P. & Penczek, P. (1998). A Ê resolution X-Ray crystallographic map of the 9A large ribosomal subunit. Cell, 93, 1105-1115. Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P. B. & Steitz, T. A. (1999). Placement of protein and Ê resolution map of the RNA structures into a 5 A 50 S ribosomal subunit. Nature, 400, 841-847. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the Ê resolution. Science, large ribosomal subunit at 2.4 A 289, 905-920. Beyer, D., Skripkin, E., Wadzack, J. & Mierhaus, K. (1994). How the ribosome moves along the mRNA during protein synthesis. J. Biol. Chem. 269, 30713-30717. Carson, M. L. (1987). Ribbon models of macromolecules. J. Mol. Graph. 5, 103-106. Carter, A., Clemons, W., Brodersen, D., Morgan-Warren, R., Wimberly, B. & Ramakrishnan, V. (2000). Functional insights from the structure of the 30 S ribosomal subunit and its interactions with antibiotics. Nature, 407, 340-348.
Movement of the Decoding Region Accompanies Translation Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N. & Noller, H. F. (1999). X-ray crystal structures of 70 S ribosome functional complexes. Science, 285, 2095-2104. Clemons, W. M., May, J. L. C., Wimberley, B. T., McCutcheon, J. P., Capel, M. S. & Ramakrishnan, V. Ê (1999). Structure of a bacterial 30 S subunit at 5.5 A resolution. Nature, 400, 833-840. Czworkowski, J., Wang, J., Steitz, T. & Moore, P. (1994). The crystal structure of elongation factor G comÊ resolution. EMBO J., 13, plexed with GDP, at 2.7 A 3661-3668. Dabrowski, M., Spahn, C. M., SchaÈfer, M. A., Patzke, S. & Nierhaus, K. H. (1998). Protection patterns of tRNAs do not change during ribosomal translocation. J. Biol. Chem. 273, 32793-32800. Davies, J. & Davis, B. D. (1968). Misreading of ribonucleic acid code words induced by aminoglycoside antibiotics. The effect of drug concentration. J Biol Chem, 243, 3312-3316. Easterwood, T. R., Major, F., Malhotra, A. & Harvey, S. C. (1994). Orientations of transfer RNA in the ribosomal A and P-sites. Nucl. Acids Res. 22, 37793786. Fourmy, D., Recht, M. I. & Blanchard, S. C. (1996). Structure of the A site of Escherichia coli 16 S ribosomal RNA complexed with an aminoglycoside antibiotic. Science, 274, 1367. Fourmy, D., Yoshizawa, S. & Puglisi, J. D. (1998). Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. J. Mol. Biol. 277, 333-345. Frank, J. & Agrawal, R. K. (2000). A ratchet-like intersubunit reorganization of the ribosome during translocation. Nature, 406, 318-322. Gabashvili, I. S., Agrawal, R. K., Spahn, C. M., Grassucci, R. A., Svergun, D. I., Frank, J. & Penczek, P. (2000). Solution structure of the E. coli Ê resolution. Cell, 100, 53770 S ribosome at 11. 5 A 549. Grajevskaja, R. A., Ivanov, Y. V. & Saminsky, E. M. (1982). 70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding. Eur. J. Biochem. 128, 47-52. Gutell, R. R. (1994). Collection of small subunit (16 Sand 16 S-like) ribosomal RNA structures: 1994. Nucl. Acids Res. 22, 3502-3507. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. ser. A, 47, 110-119. Kirillov, S. V., Makarov, E. M. & YuP, S. (1983). Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site. FEBS Letters, 157, 91-94. Lill, R., Robertson, J. M. & Wintermeyer, W. (1984). tRNA binding sites of ribosomes from Escherichia coli. Biochemistry, 23, 6710-6717. Moore, P. (1998). The three dimensional structure of the ribosome and its components. Annu. Rev. Biophys. Biomol. Struct. 27, 35-58.
515 Purohit, P. & Stern, S. (1994). Interactions of a small RNA with antibiotic and RNA ligands of the 30 S subunit. Nature, 370, 659-662. Ramakrishnan, V. & White, S. W. (1998). Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome. Trends Biochem. Sci. 23, 208-212. Rheinberger, H. J., Sternbach, H. & Nierhaus, K. H. (1981). Three tRNA binding sites on Escherichia coli ribosomes. Proc. Natl Acad. Sci. USA, 78, 5310-5314. Rodnina, M. V., Savelsbergh, A., Katunin, V. I. & Wintermeyer, W. (1997). Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature, 385, 37-41. Savelsbergh, A., Matassova, N. B., Rodnina, M. V. & Wintermeyer, W. (2000). Role of domains 4 and 5 in elongation factor G functions on the ribosome. J. Mol. Biol. 300, 951-961. Spirin, A. S. (1985). Ribosomal translocation: facts and models. Prog. Nucl. Acid Res. Mol. Biol, 32, 75-114, . Stark, H., Rodnina, M. V., Wieden, H. J., van Heel, M. & Wintermeyer, W. (2000). Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation. Cell, 100, 301-309. Sussman, J. L., Holbrook, S. R., Warrant, R. W. & Church, G. M. (1978). Crystal structure of yeast phenylalanine transfer RNA. J. Mol. Biol. 123, 607630. VanLoock, M. S., Easterwood, T. R. & Harvey, S. C. (1999). Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA-decoding site. J. Mol. Biol. 285, 2069-2078. VanLoock, M. S., Malhotra, A., Case, D. A., Agrawal, R., Penczek, P., Easterwood, T. R., Frank, J. & Harvey, S. C. (2000). A functional interpretation of the cryo-electron microscopy map of the 30 S ribosomal subunit from Escherichia coli. In The Ribosome: Structure, Function, Antibiotics and Cellular Interactions (Garrett, R. A., Douthwaite, S. R., Liljas, A., Matheson, A. T., Moore, P. B. & Noller, H. F., eds), pp. 165-171, ASM Press, Washington, DC. Wadzack, J., Burkhardt, N., Junemann, R. & Diedrich, G. (1997). Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation). J. Mol. Biol. 266, 343-356. Wilson, K. S. & Noller, H. F. (1998). Molecular movement inside the translational engine. Cell, 92, 337349. Wimberly, B., Broderson, D., Clemons, W., MorganWarren, R., Carter, A., Vonrhein, C., Hartsch, T. & Ramakrishnan, V. (2000). Structure of the 30 S ribosomal subunit. Nature, 407, 327-339. Woodson, S. A. & Leontis, N. B. (1998). Structure and dynamics of ribosomal RNA. Curr. Opin. Struct. Biol. 8, 294-300. Yoshizawa, S., Fourmy, D. & Puglisi, J. D. (1999). Recognition of the codon-anticodon helix by ribosomal RNA. Science, 285, 1722-1725.
Edited by I. Tinoco (Received 9 August 2000; received in revised form 3 October 2000; accepted 9 October 2000)
J. Mol. Biol. (2000) 304, 507±515
COMMUNICATION
Movement of the Decoding Region of the 16 S Ribosomal RNA Accompanies tRNA Translocation Margaret S. VanLoock1, Rajendra K. Agrawal2,3, Irene S. Gabashvili2 Li Qi2,3, Joachim Frank3,4 and Stephen C. Harvey1* 1
Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham, Birmingham AL 35294-0005, USA 2
Wadsworth Center
3
Department of Biomedical Sciences, State University of New York at Albany 4
Howard Hughes Medical Institute, Health Research Inc. at the Wadsworth Center Empire State Plaza, PO Box 509, Albany, NY 12201-0509, USA *Corresponding author
The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)2 complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately Ê toward the P-site. This implies that elongation factor G actively 8A pushes both the decoding site and the mRNA/tRNA complex during translocation. # 2000 Academic Press
Keywords: ribosome; elongation factor; decoding site; translation; translocation
The ribosome is the site of protein synthesis in the cell. In bacteria, it is composed of two unequal subunits: the smaller 30 S subunit, which is responsible for decoding the messenger RNA (mRNA), and the larger 50 S subunit, which catalyzes peptide bond formation. The ribosome has three principal transfer RNA (tRNA) binding sites between the subunits (Grajevskaja et al., 1982; Kirillov et al., 1983; Lill et al., 1984; Rheinberger et al., 1981): the aminoacyl site (A-site), the peptidyl site (P-site), and the exit site (E-site). Following peptide bond formation (transpeptidation), hydrolysis of GTP by elongation factor G (EF-G) is used to drive the translocation of the peptidyl tRNA from the A-site to the P-site and, simultaneously, the transport of the uncharged tRNA from the P-site to the E-site. However, translocation at substantially reduced levels can occur in the absence of EF-G (Spirin, 1985). Abbreviations used: EF-G, elongation factor G, cryoEM, cryo-electron microscopy. E-mail address of the corresponding author: [email protected] 0022-2836/00/040507±9 $35.00/0
Previous structural work on individual ribosomal proteins and some fragments of ribosomal RNAs (Agrawal & Frank, 1999; Moore, 1998; Ramakrishnan & White, 1998; Woodson & Leontis, 1998), coupled with the recent progress on crystal structure determination of the ribosomal 30 S subunit (Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000), the 50 S subunit (Ban et al., 1998, 1999, 2000), and the intact 70 S ribosome (Cate et al., 1999) from the organisms living in extreme environments (i.e. halophilic archeon and thermophilic bacteria) lead to optimism that the atomic details of some key interactions will soon be revealed. This information is supplemented by cryo-electron microscopy (cryo-EM), which has yielded lower-resolution information on various ribosomal complexes in different functional states during the translational elongation cycle, revealing important conformational changes (Agrawal et al., 1996, 1998, 1999a,b, 2000; Frank & Agrawal, 2000; Gabashvili et al., 2000; Stark et al., 2000), and providing clues to the underlying interatomic interactions. One of the major goals of ribosome research is the elucidation of the structural basis for the mechanism of EF-G-dependent tRNA trans# 2000 Academic Press
508
Movement of the Decoding Region Accompanies Translation
Movement of the Decoding Region Accompanies Translation
location. Here, we show that data from a variety of high-resolution and low-resolution experiments can be integrated into models that address this issue, using a combination of manual and automated molecular modeling methods. In the 30 S ribosomal subunit, the anticodon ends of the A and P-site tRNAs bind in the highly conserved 1400-1500 region of helix 44 in the 16 S rRNA, referred to as the decoding-site region (inset in Figure 1(a)). The decoding site, together with helix 27, the 530 loop, and 1050 region of the 16 S rRNA, allows the ribosome to distinguish between cognate and non-cognate tRNAs in the A-site. However, given that the density for helix 44 is well de®ned in a variety cryo-EM maps (Agrawal et al., 1996, 1998, 1999a,b, 2000; Frank & Agrawal, 2000; Gabashvili et al., 2000; Stark et al., 2000) and that there is an atomic structure for the A-site region of this helix (Fourmy et al., 1996, 1998), we have focused our efforts on understanding the role of helix 44 and the A-site region of the decoding site in both ribosomal ®delity (VanLoock et al., 1999) and tRNA translocation. The contributions of other regions of the 16 S rRNA in these processes will begin to be deciphered as more detailed structural information becomes available. We developed an atomic model for helix 44 of the Escherichia coli 16 S rRNA (Figure 1(b)) using the phosphate positions for this helix from the Thermus thermophilus ribosomal crystal structures (Cate et al., 1999; Clemons et al., 1999) as a guide. Adjustments were made as needed so that the helix ®ts properly into Ê resolution cryo-EM map of the E. coli the 11.5 A 70 S ribosomal initiation complex (Gabashvili et al., at the riboso2000), which contains fMet-tRNAMet f mal P-site, and to accommodate sequence and secondary structure differences in this region between E. coli and T. thermophilus (Gutell, 1994). In the modeling we paid particular attention to the structure of the critical A-site region at the top of helix 44 (Figure 1(c)), which has been studied extensively. An RNA oligomer with a sequence identical to the E. coli A-site region shows tRNAdependent footprinting patterns very similar to those for the A-site in the intact ribosome (Purohit & Stern, 1994), and the structure of a similar oligo-
509 mer alone and complexed with the antibiotic paromomycin has been determined by NMR (Fourmy et al., 1996, 1998). The backbone conformation of the A-site region has been determined crystallographically, for both the isolated 30 S subunit (Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000) and for the intact 70 S ribosome with bound tRNA (Cate et al., 1999). Phosphate positions in both NMR structures (Fourmy et al., 1996, 1998) and the crystal structures (Cate et al., 1999; Clemons et al., 1999; Carter et al., 2000; Wimberly et al., 2000) are all very similar. In addition, a previously published model of the A-site region of the 16 S rRNA complexed with A and P-site tRNAs and mRNA (VanLoock et al., 1999) agrees very well with the experimentally determined structures, with a rootÊ for mean-square deviation (RMSD) of less than 1 A the phosphate positions. Interestingly, the calcuÊ resolution electron density map of the lated 7.8 A previous decoding site model (VanLoock et al., 1999) is strikingly similar to that reported for the Ê resolution 70 S ribosomal crystal structure 7.8 A (Cate et al., 1999), with a substantial hole (see Figure 6 by Cate et al., 1999) around nucleotides A1492 and A1493 in both electron density maps. Here, we used an NMR structure for an A-site RNA oligomer in the absence of aminoglycoside (Fourmy et al., 1998) to analyze conformational changes in the decoding site upon tRNA translocation. Ê We docked the model for helix 44 into the 17 A resolution cryo-EM map of the 70 S ribosome in a pre-translocation complex containing both A and P-site tRNAs and mRNA. This new three-dimensional reconstruction was obtained from a complex at the that was prepared by binding tRNAMet f P-site and Phe-tRNAPhe at the A-site of an MF-mRNA programmed ribosome (Gabashvili et al., 2000) (R.K.A, L.Q & J.F., unpublished results). No adjustments to the position of helix 44 were necessary, suggesting that its conformation is very similar to that in the initiation complex. In the pre-translocational 70 S (tRNA)2 complex, the density for the A and P-site tRNAs is very clear (Figure 2(a) and (b)). We docked the X-ray structure (Sussman et al., 1978) of tRNAPhe into the P-site tRNA density and a model for the
Figure 1. The structure of the decoding site region of the ribosome. (a) Secondary structure diagram of the E. coli 16 S rRNA (Gutell, 1994) with helix 44 highlighted in cyan, and the sequence of the decoding site region enlarged in Ê resolution, of the 30 S ribosomal subunit the inset. (b) Ribbons image (Carson, 1987) of the cryo-EM map, at 11.5 A isolated from the E. coli 70 S ribosomal initiation complex (Gabashvili et al., 2000). Helix 44 is highlighted in cyan and the placement of the atomic model for the entire helix 44 is shown in purple. The black box indicates the decoding-site region of helix 44. The EM map is shown at higher threshold values to emphasize the shape and location of the helix 44. At lower threshold values, the entire structure of helix 44 is surrounded by density. (c) The region has been enlarged and displayed in stereo to show the structural details. The structure displayed in the electron density corresponding to the A-site was taken from an NMR structure of an RNA oligomer mimicking the A-site region of the decoding site (Fourmy et al., 1998). The phosphate positions in this structure are very similar to that of both a model for the decoding site in a complex with A and P-site tRNAs and mRNA and the X-ray crystal structures of the isolated 30 S subunit (Clemons et al., 1999) and the intact 70 S ribosome with bound tRNA (Cate et al., 1999). The enlarged area is the primary focus of this article, and the remaining Figures involving the A-site structure are shown from this view.
510
Movement of the Decoding Region Accompanies Translation
Figure 2. Stereo view showing the ®t of an NMR structure of an A-site RNA oligomer (Fourmy et al., 1998) in the cryo-EM density. (a) Fitting of the A-site region of the decoding site (blue) into the cryo-EM density of the pre-translocation 70 S(tRNA)2 complex (grey). Density corresponding to the P-site tRNA (green) is also visible in this ribosomal reconstruction. The EM density is shown at higher threshold values for clarity. At lower threshold levels, the atomic structure of the tRNAs is fully covered by EM density. (b) Same view as in (a), with a codon of mRNA (cyan) added, but without the EM density map displayed. The A-site mRNA-tRNA complex lies on the major-groove side of the decodingsite helix in close proximity to the highly conserved nucleotides A1492 and A1493. These nucleotides are thought to be important for mRNA proofreading and ribosomal ®delity (Fourmy et al., 1996; VanLoock et al., 1999). Landmarks: A, A-site tRNA; P, P-site tRNA.
A-site tRNA complexed with a three-nucleotide poly(U)mRNA (Easterwood et al., 1994) into the density corresponding to the A-site tRNA. The orientation of the A and P-site tRNAs is very simi-
lar to that reported on in the X-ray crystal structure of the 70 S ribosome (Cate et al., 1999), with only slight variations in the A-site tRNA position. In this complex, the A-site codon-anticodon complex
Movement of the Decoding Region Accompanies Translation
lies on the major groove side of the decoding site helix, in close proximity to the universally conserved nucleotides A1492 and A1493 (Figure 2(b)). This is consistent with previous studies suggesting that these nucleotides may interact directly with the mRNA in the decoding process, and that this interaction may contribute to translational ®delity (Fourmy et al., 1996; VanLoock et al., 1999). The importance of these nucleotides is indicated by the ®nding that mutation or deletion of A1492 and A1493 is lethal to the cell (Yoshizawa et al., 1999). We analyzed the A-site region of the decoding site from cryo-EM density maps of the 70 S ribosome in the pre-translocational state with EF-G and a non-hydrolyzable GTP analog with bound A and P-site tRNAs (Agrawal et al., 1999a), the 70 S (tRNA)2 EF-G GMPPCP complex, and in a complex with EF-G GDP and fusidic acid with bound P and E-site tRNAs (Agrawal et al., 1999a), the 70 S (tRNA)2 EF-G GDP fusidic acid complex. These cryo-EM maps provide a snapshot of the ribosome immediately after peptide bond formation (pre-translocation complex), upon binding of EFG-GTP to the ribosome (70 S (tRNA)2 EFG GMPPCP complex), and just after tRNA translocation, but prior to EF-G dissociation from the ribosome (70S (tRNA)2 EF-G GDP fusidic acid complex). We docked the model for helix 44 into the cryo-EM maps. The lower two-thirds of helix 44 ®ts very nicely into all of these maps. The A-site region of helix 44 ®ts well into the density for the 70 S (tRNA)2 EF-G GMPPCP complex but poorly into the density for the 70 S (tRNA)2 EFG GDP fusidic acid complex. We made the appropriate changes necessary to ®t the model into the electron density map for the latter state, to understand better the nature of the conformational changes associated with tRNA translocation. The density corresponding to the A-site region of the decoding site moves toward the P-site region Ê when comparing the by approximately 8 A 70 S EF-G GDP fusidic acid complex to the pretranslocation and 70 S EF-G GMPPCP complexes (Figure 3(a)). In order to accommodate this change, the turn in the upper part of helix 44 around nucleotides 1410-1490 rotates slightly, and the entire decoding-site structure shifts toward Ê the P-site tRNA-binding site by approximately 8 A (Figure 3(b) and (c)). As a result of the conformational change, nucleotides A1492 and A1493 Ê toward the P-site move by approximately 12 A tRNA-binding region (Figure 3(c)). This shift corresponds roughly to three nucleotides or one codon. Similar shifts in the P-site region of the decoding site density are also visible, but since its structure is not well de®ned, the exact nature of the shifts is unclear. Previous studies have suggested that the mass center of the A and P-site tRNAs moves Ê during translocation by approximately 12 A (Wadzack et al., 1997), and that the mRNA is shifted by exactly one codon (Beyer et al., 1994). The current results thus suggest that during translocation, the decoding site region moves in the
511 same direction and by approximately the same amount as the A and P-site tRNAs. In order to understand the role of EF-G in translocation, we displayed the previously (Agrawal et al., 1999a) ®tted X-ray structure of EF-G (Czworkowski et al., 1994) in the cryo-EM map of the 70 S EF-G GDP fusidic acid complex (Figure 3(b)). In this complex, domain IV of EF-G binds in the region that was occupied by the A-site tRNA anticodon stem loop in the pre-translocation complex (compare Figure 2(a) with 3(b): see also Agrawal et al., 1999a). In addition, the tip of EF-G domain IV lies in a patch of the ribosomal molecular envelope previously occupied by the A-site region of helix 44 in the pre-translocation complex (Figures 2(a) and 3(b)). It is known that amino acid mutations or deletions in the tip of domain IV signi®cantly decrease the rate of tRNA translocation (Savelsbergh et al., 2000), suggesting that it is critical for EF-G function. The location of EF-G domain IV in the 70 S EF-GGDP fusidic acid complex is highly suggestive of its function. During translocation, domain IV may actively push both the decoding site and the mRNA-tRNA complex from the A-site to the P-site. The tip of EF-G domain IV may also serve as a placeholder by occupying the A site and preventing slippage of the mRNAtRNA complex back into the A-site before translocation is complete. Both of these mechanisms would be consistent with the ®nding that deletion of EF-G domain IV reduces the rate of translocation 1000-fold, without affecting EF-G binding or GTP hydrolysis (Rodnina et al., 1997). Once EF-G dissociates from the ribosome, the decoding site is free to move back to its original position and accept a new incoming A-site tRNA. The back-and-forth movement of the decoding site region during translocation is consistent with suggestions that the decoding site acts as a molecular ratchet that carries the mRNA through the 30 S mRNA channel (VanLoock et al., 2000). Indeed, recent cryo-EM studies have shown that the entrance to the mRNA channel in the 30 S subunit opens following EF-G GTP binding, and that, during translocation, the 30 S subunit undergoes a ratchet-like rotation that follows the direction of tRNA movement relative to the 50 S subunit (Frank & Agrawal, 2000). Together, these data suggest that the ribosome actively participates in the translocation of the mRNA-tRNA complex from one tRNA binding site to another, and that the observed conformational changes seen in the tRNA-binding regions of the 30 S subunit consistently follow the direction of tRNA movement in the ribosome. Ê shift of the decoding region of helix 44 The 8 A during translocation may be necessary to help lower the energy barrier associated with tRNA translocation. The energy freed up by EF-G-dependent GTP hydrolysis is used to induce a conformational change in the A-site that may serve to position the A-site tRNA such that it can ef®ciently move into the P-site and, therefore, lower the
512
Movement of the Decoding Region Accompanies Translation
Figure 3 (legend opposite)
Movement of the Decoding Region Accompanies Translation
513
Figure 4. Stereo view of superimposed models of helix 44 in the pre and post-translocation complexes. The pretranslocation 70 S(tRNA)2 complex is shown in blue and the 70 S (tRNA)2 EF-G GDP fusidic acid complex in red (Agrawal et al., 1999a). The lower portion of helix 44 remains ®xed while its tip, the decoding region, undergoes signi®cant conformational changes upon EF-G-dependent GTP hydrolysis and tRNA translocation. The arrow indicates the direction of the movement of the A (pink) and P-site (green) tRNAs, the mRNA (cyan), and the decoding region during tRNA translocation.
Figure 3. Stereo view of the cryo-EM densities from the decoding site region of the pre and post-translocation complexes. (a) Comparison of the density for the A-site region of helix 44 from cryo-EM maps of the pre-translocation 70 S (tRNA)2 complex (black) and the 70 S (tRNA)2 EF-G GDP fusidic acid complex (Agrawal et al., 1999a) (cyan). There is a signi®cant shift in the electron density for the A-site region of helix 44 toward the P-site tRNA-binding domain upon EF-G-dependent GTP hydrolysis. (b) Fitting of the structure for the A-site region of the 16 S rRNA decoding site (Fourmy et al., 1998) (red) into the cryo-EM map (cyan) of the 70 S (tRNA)2 EF-G GDP-fusidic acid complex. The structure of the decoding site as shown in Figure 2 was altered from that in the pre-translocation comÊ towards the P-site. A major portion of domain IV of EF-G (yellow) plex and was shifted by approximately 8 A occupies the same position occupied previously by the A-site tRNA in the pre-translocation complex (see Agrawal et al., 1999a) (Figure 2(a)), and the tip of domain IV lies in the electron density previously occupied by the 16 S rRNA decoding-site region in both the pre-translocational and the 70 S (tRNA)2 EF-G GMPPCP complexes. In this reconstruction, the A-site tRNA (pink) seen in the pre-translocation complex (Figure 2) has been moved to the P-site upon EF-G-dependent GTP hydrolysis. (c) Comparison of the decoding-site structures from the pre-translocation complex (blue) and the 70 S (tRNA)2 EF-G GDP fusidic acid complex (red). In this transition, the A-site region shifts about Ê toward the P-site tRNA-binding domain, and rotates slightly. As a consequence of these combined motions, 8A Ê toward the P-site, roughly the length of one codon. A1492 and A1493 are moved approximately 12 A
514 activation energy barrier of tRNA translocation. In the absence of EF-G the decoding site may fail to undergo the observed conformational changes. However, some small fraction of tRNAs may still overcome the energy barrier associated with translocation and actively translocate on their own, a phenomenon that has been observed experimentally (Spirin, 1985). The observed movement in the A-site region of the decoding site is in line with previous suggestions that the ribosome has a moveable domain (Dabrowski et al., 1998) that drives translocation of tRNA. The lower portion of helix 44, which remains motionless throughout translocation (Figure 4), may serve as an anchoring point for the decoding site region. During translocation, EF-G binds to the ribosome and its GTP-binding domain interacts with the a-sarcin-ricin loop on the 50 S subunit (Agrawal et al., 1999a). Upon GTP hydrolysis, the a-sarcin-ricin loop may undergo a conformational change that alters its interactions with the large subunit protein L14 which forms an intersubunit contact with helix 44 of the 30 S subunit (Gabashvili et al., 2000). These conformational changes may in turn affect the conformation of the decoding site. A second contact between the decoding site and the 1920 region of the 50 S subunit, referred to as bridge B2a (Gabashvili et al., 2000), might also be involved in the binding of EF-G and tRNA translocation. The density around this contact changes upon EF-G-dependent GTP hydrolysis, and nucleotides in this region of the 50 S are protected from chemical attack in the presence of EF-G GDP and fusidic acid (Wilson & Noller, 1998). Conformational changes in the A-site during translocation would also help explain the decreased tRNA translocation rate in the presence of aminoglycoside antibiotics (Davies & Davis, 1968). Binding of aminoglycosides to the decoding site may lock the A-site in the conformation observed before EF-Gdependent GTP hydrolysis, therefore preventing structural changes in this region which are necessary for ef®cient tRNA translocation. Taken together, these results strongly suggest that, after transpeptidation, the energy from EF-Gdependent GTP hydrolysis is used by the ribosome to actively push both the decoding site and the mRNA-tRNA complex during translocation. This movement is consistent with the proposal that the ribosome is acting as a molecular ratchet (Frank & Agrawal, 2000; VanLoock et al., 2000). Materials and methods Atomic models for helix 44 were developed manually using a combination of molecular modeling packages including Insight II, Quanta (both products of Molecular Simulations Inc.), and O (Jones et al., 1991). Coordinates for the models will be made available from http://uracil.cmc.uab.edu/Publications. Coordinates for the P-site tRNA were taken from the X-ray structure of tRNAPhe (Sussman et al., 1978). The
Movement of the Decoding Region Accompanies Translation tRNAs were docked into their corresponding binding sites in each of the ribosomal cryo-EM reconstructions. Coordinates for the A-site tRNA and mRNA were taken from a previously published model of the A-site tRNA/ mRNA complex (Easterwood et al., 1994).
Acknowledgments The authors thank Scott Stagg, Christian Spahn and Pawel Penczek for helpful discussions. The work was supported by grants from the National institutes of Health: R01 GM53827 (S.C.H.), R37 GM29169 (J.F.), and R01 GM 55440 (J.F.).
References Agrawal, R. K. & Frank, J. (1999). Structural studies of the translational apparatus. Curr. Opin. Struct. Biol. 9, 215-221. Agrawal, R. K., Penczek, P., Grassucci, R. A., Li, Y., Leith, A., Nierhaus, K. H. & Frank, J. (1996). Direct visualization of A, P, and E-site transfer RNAs in the Escherichia coli ribosome. Science, 271, 1000-1002. Agrawal, R. K., Penczek, P., Grassucci, R. A. & Frank, J. (1998). Visualization of elongation factor G on the Escherichia coli 70 S ribosome: the mechanism of translocation. Proc. Natl Acad. Sci. USA, 95, 61346138. Agrawal, R. K., Heagle, A. B., Penczek, P., Grassucci, R. A. & Frank, J. (1999a). EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70 S ribosome. Nature Struct. Biol. 6, 643-647. Agrawal, R. K., Lata, R. K. & Frank, J. (1999b). Conformational variability in Escherichia coli 70 S ribosome as revealed by 3D cryo-electron microscopy. Int. J. Biochem. Cell Biol. 31, 243-254. Agrawal, R. K., Heagle, A. B. & Frank, J. (2000). Studies of elongation factor G-dependent tRNA translocation by three-dimensional cryo-electron microscopy. In The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions (Garrett, R. A., Douthwaite, S. R., Liljas, A., Matheson, A. T. & Moore, P. B., eds), pp. 53-62, ASM Press, Washington, DC. Ban, N., Freeborn, B., Nissen, P. & Penczek, P. (1998). A Ê resolution X-Ray crystallographic map of the 9A large ribosomal subunit. Cell, 93, 1105-1115. Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P. B. & Steitz, T. A. (1999). Placement of protein and Ê resolution map of the RNA structures into a 5 A 50 S ribosomal subunit. Nature, 400, 841-847. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the Ê resolution. Science, large ribosomal subunit at 2.4 A 289, 905-920. Beyer, D., Skripkin, E., Wadzack, J. & Mierhaus, K. (1994). How the ribosome moves along the mRNA during protein synthesis. J. Biol. Chem. 269, 30713-30717. Carson, M. L. (1987). Ribbon models of macromolecules. J. Mol. Graph. 5, 103-106. Carter, A., Clemons, W., Brodersen, D., Morgan-Warren, R., Wimberly, B. & Ramakrishnan, V. (2000). Functional insights from the structure of the 30 S ribosomal subunit and its interactions with antibiotics. Nature, 407, 340-348.
Movement of the Decoding Region Accompanies Translation Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N. & Noller, H. F. (1999). X-ray crystal structures of 70 S ribosome functional complexes. Science, 285, 2095-2104. Clemons, W. M., May, J. L. C., Wimberley, B. T., McCutcheon, J. P., Capel, M. S. & Ramakrishnan, V. Ê (1999). Structure of a bacterial 30 S subunit at 5.5 A resolution. Nature, 400, 833-840. Czworkowski, J., Wang, J., Steitz, T. & Moore, P. (1994). The crystal structure of elongation factor G comÊ resolution. EMBO J., 13, plexed with GDP, at 2.7 A 3661-3668. Dabrowski, M., Spahn, C. M., SchaÈfer, M. A., Patzke, S. & Nierhaus, K. H. (1998). Protection patterns of tRNAs do not change during ribosomal translocation. J. Biol. Chem. 273, 32793-32800. Davies, J. & Davis, B. D. (1968). Misreading of ribonucleic acid code words induced by aminoglycoside antibiotics. The effect of drug concentration. J Biol Chem, 243, 3312-3316. Easterwood, T. R., Major, F., Malhotra, A. & Harvey, S. C. (1994). Orientations of transfer RNA in the ribosomal A and P-sites. Nucl. Acids Res. 22, 37793786. Fourmy, D., Recht, M. I. & Blanchard, S. C. (1996). Structure of the A site of Escherichia coli 16 S ribosomal RNA complexed with an aminoglycoside antibiotic. Science, 274, 1367. Fourmy, D., Yoshizawa, S. & Puglisi, J. D. (1998). Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. J. Mol. Biol. 277, 333-345. Frank, J. & Agrawal, R. K. (2000). A ratchet-like intersubunit reorganization of the ribosome during translocation. Nature, 406, 318-322. Gabashvili, I. S., Agrawal, R. K., Spahn, C. M., Grassucci, R. A., Svergun, D. I., Frank, J. & Penczek, P. (2000). Solution structure of the E. coli Ê resolution. Cell, 100, 53770 S ribosome at 11. 5 A 549. Grajevskaja, R. A., Ivanov, Y. V. & Saminsky, E. M. (1982). 70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding. Eur. J. Biochem. 128, 47-52. Gutell, R. R. (1994). Collection of small subunit (16 Sand 16 S-like) ribosomal RNA structures: 1994. Nucl. Acids Res. 22, 3502-3507. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. ser. A, 47, 110-119. Kirillov, S. V., Makarov, E. M. & YuP, S. (1983). Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site. FEBS Letters, 157, 91-94. Lill, R., Robertson, J. M. & Wintermeyer, W. (1984). tRNA binding sites of ribosomes from Escherichia coli. Biochemistry, 23, 6710-6717. Moore, P. (1998). The three dimensional structure of the ribosome and its components. Annu. Rev. Biophys. Biomol. Struct. 27, 35-58.
515 Purohit, P. & Stern, S. (1994). Interactions of a small RNA with antibiotic and RNA ligands of the 30 S subunit. Nature, 370, 659-662. Ramakrishnan, V. & White, S. W. (1998). Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome. Trends Biochem. Sci. 23, 208-212. Rheinberger, H. J., Sternbach, H. & Nierhaus, K. H. (1981). Three tRNA binding sites on Escherichia coli ribosomes. Proc. Natl Acad. Sci. USA, 78, 5310-5314. Rodnina, M. V., Savelsbergh, A., Katunin, V. I. & Wintermeyer, W. (1997). Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature, 385, 37-41. Savelsbergh, A., Matassova, N. B., Rodnina, M. V. & Wintermeyer, W. (2000). Role of domains 4 and 5 in elongation factor G functions on the ribosome. J. Mol. Biol. 300, 951-961. Spirin, A. S. (1985). Ribosomal translocation: facts and models. Prog. Nucl. Acid Res. Mol. Biol, 32, 75-114, . Stark, H., Rodnina, M. V., Wieden, H. J., van Heel, M. & Wintermeyer, W. (2000). Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation. Cell, 100, 301-309. Sussman, J. L., Holbrook, S. R., Warrant, R. W. & Church, G. M. (1978). Crystal structure of yeast phenylalanine transfer RNA. J. Mol. Biol. 123, 607630. VanLoock, M. S., Easterwood, T. R. & Harvey, S. C. (1999). Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA-decoding site. J. Mol. Biol. 285, 2069-2078. VanLoock, M. S., Malhotra, A., Case, D. A., Agrawal, R., Penczek, P., Easterwood, T. R., Frank, J. & Harvey, S. C. (2000). A functional interpretation of the cryo-electron microscopy map of the 30 S ribosomal subunit from Escherichia coli. In The Ribosome: Structure, Function, Antibiotics and Cellular Interactions (Garrett, R. A., Douthwaite, S. R., Liljas, A., Matheson, A. T., Moore, P. B. & Noller, H. F., eds), pp. 165-171, ASM Press, Washington, DC. Wadzack, J., Burkhardt, N., Junemann, R. & Diedrich, G. (1997). Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation). J. Mol. Biol. 266, 343-356. Wilson, K. S. & Noller, H. F. (1998). Molecular movement inside the translational engine. Cell, 92, 337349. Wimberly, B., Broderson, D., Clemons, W., MorganWarren, R., Carter, A., Vonrhein, C., Hartsch, T. & Ramakrishnan, V. (2000). Structure of the 30 S ribosomal subunit. Nature, 407, 327-339. Woodson, S. A. & Leontis, N. B. (1998). Structure and dynamics of ribosomal RNA. Curr. Opin. Struct. Biol. 8, 294-300. Yoshizawa, S., Fourmy, D. & Puglisi, J. D. (1999). Recognition of the codon-anticodon helix by ribosomal RNA. Science, 285, 1722-1725.
Edited by I. Tinoco (Received 9 August 2000; received in revised form 3 October 2000; accepted 9 October 2000)