Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process
2002; Springer Nature; Volume: 21; Issue: 13 Linguagem: Inglês
10.1093/emboj/cdf326
ISSN1460-2075
AutoresMikel Valle, Jayati Sengupta, N.K. Swami, Robert A. Grassucci, Nils Burkhardt, Knud H. Nierhaus, Rajendra K. Agrawal, Joachim Frank,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle1 July 2002free access Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process Mikel Valle Mikel Valle Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Jayati Sengupta Jayati Sengupta Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Neil K. Swami Neil K. Swami Columbia High School, 962 Luther Road, East Greenbush, NY, 12061 USA Search for more papers by this author Robert A. Grassucci Robert A. Grassucci Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Nils Burkhardt Nils Burkhardt Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Knud H. Nierhaus Knud H. Nierhaus Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Rajendra K. Agrawal Rajendra K. Agrawal Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Mikel Valle Mikel Valle Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Jayati Sengupta Jayati Sengupta Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Neil K. Swami Neil K. Swami Columbia High School, 962 Luther Road, East Greenbush, NY, 12061 USA Search for more papers by this author Robert A. Grassucci Robert A. Grassucci Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Nils Burkhardt Nils Burkhardt Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Knud H. Nierhaus Knud H. Nierhaus Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Rajendra K. Agrawal Rajendra K. Agrawal Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Author Information Mikel Valle1, Jayati Sengupta2, Neil K. Swami3, Robert A. Grassucci1, Nils Burkhardt4, Knud H. Nierhaus4, Rajendra K. Agrawal2,5 and Joachim Frank 1,5 1Howard Hughes Medical Institute, Health Research, Inc., at the New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA 2Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY, 12201-0509 USA 3Columbia High School, 962 Luther Road, East Greenbush, NY, 12061 USA 4Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany 5Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3557-3567https://doi.org/10.1093/emboj/cdf326 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon–anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition. Introduction In protein biosynthesis the decoding process is the fundamental step that assures the accuracy of the translation from mRNA into polypeptide. The codon in the mRNA has to be correctly recognized by the anticodon of the cognate tRNA, leading to its incorporation, while incorrect matches must lead to rejection. Elongation factor Tu (EF-Tu) plays a crucial role in this function of delivering the aminoacyl-tRNA (aa-tRNA) as part of the ternary complex (aa-tRNA·EF-Tu·GTP). When this ternary complex binds to the ribosome, the tRNA is bound in the A/T state, as its anticodon is able to interact with the mRNA in the A-site while its acceptor end is still bound to the factor (Moazed and Noller, 1989). In this way, the codon–anticodon match can be tested before the new amino acid is incorporated. When a cognate tRNA is present, EF-Tu catalyzes the hydrolysis of GTP and changes its conformation (Berchtold et al., 1993; Kjeldgaard et al., 1993; Polekhina et al., 1996), thereby reducing its affinity for the ribosome and the tRNA (Dell et al., 1990). The remaining EF-Tu·GDP binary complex leaves the ribosome, so that the tRNA is free to move its CCA end towards the peptidyltransferase center (PTC), allowing the polypeptide to be elongated by the new amino acid residue (Kaziro, 1978). The transition of tRNA from the A/T site to A-site is known as accommodation. Several important questions have remained unanswered. First, how the anticodon of the aa-tRNA recognizes the codon in the mRNA while it is delivered by EF-Tu at a very different angle compared with the orientation assumed in the final A-site. Secondly, how this codon–anticodon recognition acts as a signal to trigger the GTP hydrolysis and the release of EF-Tu. And finally, what mechanism is employed that makes possible a tRNA rotation towards the PTC while maintaining the codon–anticodon pairing. As a signal for GTP hydrolysis, one possibility is that the cognate tRNA–mRNA interaction in the decoding center promotes conformational changes in the ribosome (Geigenmüller and Nierhaus, 1990; Powers and Noller, 1994; Lodmell and Dahlberg, 1997) that are communicated through the inter-subunit bridges to the GTPase-associated center of the 50S subunit (Rodnina and Wintermeyer, 2001). Another possibility that has been discussed is that a conformational change in the tRNA following codon–anticodon pairing (Moras et al., 1985, 1986) might act as the signal (Rodnina et al., 1994; Yarus and Smith, 1995). The ribosome-bound EF-Tu ternary complex has previously been visualized by cryo-electron microscopy (EM) techniques in the presence of the antibiotic kirromycin (Stark et al., 1997; Agrawal et al., 2000b). Kirromycin is known to block the conformational changes in EF-Tu that follow GTP hydrolysis (Wolf et al., 1977; Parmeggiani and Stewart, 1985). As a result of this inhibition, the factor remains bound to the ribosome in a conformation that is thought to be an intermediate between the GTP and the GDP states. This previous study suggested that EF-Tu binds to the base of L7/L12 stalk of the 50S subunit, close to the universally conserved α-sarcin–ricin loop (SRL) of the 23S rRNA, and showed the anticodon loop of the tRNA interacting with the decoding region. One of the puzzles posed by the results of previous cryo-EM studies is that the orientation of the tRNA deduced does not facilitate codon–anticodon interaction. In the present work we have employed cryo-EM to study a complex of 70S ribosomes with P-site tRNA and EF-Tu ternary complex carrying cognate aa-tRNA and GDP in the presence of kirromycin. The first three-dimensional (3D) reconstruction of the whole set of single particles (see Agrawal et al., 2000b) showed low (∼50%) density in the region of the ternary complex. Computation of a difference map revealed the overall binding position of the complex, but a detailed interpretation of binding sites was not possible. We have now applied a supervised classification strategy based on the similarity of these images with distinct 3D references. One of the resultant 3D maps shows the ternary complex with much higher definition than seen in the earlier reports (Stark et al., 1997; Agrawal et al., 2000b). Most importantly, EF-Tu and the tRNA are now distinctly recognizable, and the tRNA is seen to interact with the base of the L7/L12 stalk and with protein S12. Detailed fitting, supported by quantitative measures, reveals that the tRNA structure is different from the reported X-ray structures and from the structure deduced in the former cryo-EM work (Stark et al., 1997) due to a change of orientation in the anticodon arm, towards an orientation that facilitates codon–anticodon interaction. Furthermore, the T-loop side of the tRNA is seen to interact with the 58-nucleotide segment of the 23S rRNA that forms a complex with protein L11 (Wimberly et al., 1999), a region known as the GTPase-associated center (GAC). The results bring new insights in understanding the incorporation of tRNA into the ribosome and strongly implicate tRNA as an active player in this process. Results 3D reconstruction and analysis of the cryo-EM maps A set of 22 905 projections was selected by a previously described quasi-automated method (Lata et al., 1995). By image processing techniques (see Materials and methods), a first map was calculated. The map showed the presence of extra density located in the inter-subunit space, which we attributed to the ternary complex. However, the extra density was fragmented, and could only be seen by rendering the volume at lowered density threshold. This initial poor representation of the putative ternary complex can be understood either as the result of averaging a population of ribosomes with low occupancy of the factor, or as a consequence of merging particles where the complex exists in different conformations, or as a combination of both effects. Evidence that low occupancy is a major factor comes from the filter-binding assay (see Materials and methods), which estimated ∼50% occupancy for the ternary complex. In order to separate the heterogeneous particle set into more homogeneous subsets, we have used a supervised classification method (details described in the Supplementary data, available at The EMBO Journal Online), where the images are classified according to their similarity to distinct references (see Frank, 1996). Briefly, our strategy for separation makes use of the resemblance that the ternary complex and EF-G share in their architecture and their overall binding position on the ribosome, and the fact that the projection of ribosomes carrying the ternary complex would show more similarity with an EF-G bound ribosome than with a ribosome with an empty A-site. After the selection, new 3D reconstructions were computed. Application of the same method of classification to a control set of empty ribosomes did not show a mass at the A-site in the reconstruction from the subset having highest similarity with the EF-G bound reference (see Supplementary data). The final 3D cryo-maps are depicted in Figure 1A (map 1) and B (map 2). Final map 1 (Figure 1A) was reconstructed from particles more similar to an empty ribosome. It is clear that the ternary complex occupancy is poor. In contrast, the occupancy is large in final map 2 (Figure 1B), a reconstruction obtained from those particles most similar to an EF-G-bound ribosome. In this latter map (Figure 1B) the extra density (in red) is a continuous mass with a volume large enough to accommodate the whole ternary complex. Figure 1.Cryo-EM maps resulting from the classification, and their analysis. Map 1 (A, C and E) and map 2 (B, D and F) are depicted in two orientations related by a 90° rotation around a vertical axis in the plane [(A–D) show the side views, (E) and (F) the top views of the 70S ribosome). Side views are rotated in the plane by 90° from their conventional presentation (see Supplementary figure S1) to maintain continuity in the tRNA positions with (E) and (F)]. In (C)–(F) the ribosomal subunits are represented as semi-transparent surface (blue for the 50S and yellow for the 30S subunit), so that the positions of P- and E-site tRNAs (purple) and the mass attributable to the ternary complex (red) can be seen more clearly. (G) Representation of the difference map (see Materials and methods) calculated for the cryo-EM map 1, shown in green. Lobe of mass labeled with an asterisk is attributed to a conformational change in the L1 region. The ribosome is shown in same orientation as in (C). Landmarks are as follows: CP, central protuberance; Sb, L7/L12 stalk base; sp, spur; b, body; h, head; dc, decoding center; L1, stalk of L1 protein. Labels in tRNA positions: T, T-site within the ternary complex; A, aminoacyl site; P, peptidyl site; E, exit site. Download figure Download PowerPoint We isolated the densities corresponding to tRNAs and tRNA–EF-Tu complex bound to the ribosome (see Materials and methods). This separation shows that both the maps carry P- and E-site tRNAs (purple in Figure 1), and the main difference relates to the presence of the ternary complex (red). [It should be noted that in all tRNA binding experiments (e.g. Cate et al., 1999; Agrawal et al., 2000a), a mass of density corresponding to the E-site tRNA is always observed.] In map 1 (Figure 1A, C and E) the putative ternary complex is under-represented and only few parts of the tRNA and the EF-Tu can be seen. Some density clearly appears at the expected binding site of the anticodon loop of the A-site tRNA on the 30S subunit, but the rest of the tRNA is not visible directly in the volume. In a calculated difference map (see Materials and methods) for this reconstruction (in green in Figure 1G) no major difference in the ribosome conformation is detected, but there is a large mass that covers the position of an A-site tRNA reaching from the decoding site in the 30S subunit to the PTC in the 50S subunit. The region that should correspond to the CCA end of the tRNA extends below the base of L7/L12 stalk, toward the region that EF-Tu binds to. In map 2 (Figure 1B, D and F) the picture of the ternary complex (in red) changes drastically, and its full mass is now represented, together with the masses of P- and E-site tRNAs (purple). The anticodon arm of the tRNA is situated in the decoding region and the acceptor arm is still bound to the factor. No density attributable to an acceptor arm connected to the A-site in the 50S subunit is visible. In this reconstruction, both tRNA and protein interact with the base of the L7/L12 stalk, where contact points important for the activity of the elongation factors are located. The interactions between the ternary complex and the ribosome have been studied using this map. Views of the mass attributable to the ternary complex from the solvent (Figure 2A) and from side of the inter-subunit space (Figure 2B) reveal a structure distinct enough to permit visual identification of tRNA and the domains of the protein factor. To understand the nature of the elements within the complex, the X-ray structure of the Phe-tRNAPhe·EF-Tu·GTP analog complex from Thermus aquaticus (Nissen et al., 1995) was filtered to the same resolution and displayed in similar orientation (Figure 2C and D). The tRNA and EF-Tu were colored separately for the purpose of clarity. We note that the structures come from different organisms (EF-Tu from T.aquaticus and from Escherichia coli share 68% sequence identity), the complexes have different guanine nucleotides bound, and while our cryo-EM map shows the ternary complex bound to the ribosome, the X-ray structure represents the complex in the close packing of a 3D crystal. Nevertheless, the overall mass distribution and the shape of the ribosome-bound ternary complex in our cryo-EM map and in the ternary complex crystal structure are strikingly similar. Figure 2.Comparison of cryo-EM density and X-ray structure of the ternary complex. (A and B) Ternary complex density, isolated from the cryo-EM map: (A) as seen from the solvent side; (B) as seen from the intersubunit space side. (C and D) Equivalent orientations of the X-ray crystal structure from T.aquaticus ternary complex (Nissen et al., 1995), filtered to the resolution of the cryo-EM map. EF-Tu is shown in green, aa-tRNA in yellow. (E and F) Ribbons representation of the fitting of the ternary complex crystal structure into the cryo-EM density. (G–I) EF-Tu from T.aquaticus in the GTP (Kjeldgaard et al., 1993) and GDP (Polekhina et al., 1996) states are shown next to the EF-Tu·GDP·kirromycin inferred from the cryo-EM map at the same resolution. Download figure Download PowerPoint The result of the rigid-body fitting of the X-ray structure form of the ternary complex into the cryo-EM map is presented in Figure 2E and F. Clearly, the domains of the elongation factor have not undergone a large rearrangement in the interaction with the ribosome, but the tRNA has. In the cryo-EM map, the acceptor arm of the tRNA that is held by EF-Tu apparently maintains its binding with the protein as in the crystal structure, but the elbow of the tRNA has moved significantly from its position in the crystal structure. However, in both versions of the tRNA, the anticodon loops are in the same position due to an apparent kink in the anticodon arm. In this way, the T and D loops forming the elbow of the tRNA in our cryo-EM map move toward the base of the L7/L12 stalk of the ribosome, while the two ends of the L-shaped structure remain in the same place, changes that imply a large internal reorganization of the tRNA structure. Docking of tRNA and EF-Tu X-ray structures The E.coli EF-Tu has been divided into three domains: domain I, the guanine nucleotide binding domain that accounts for the first 204 amino acids of the N-terminus; domain II, residues 205–298; and domain III, the C-terminal domain that covers positions 299–393. In the X-ray structure of T.aquaticus EF-Tu, the constellation of these domains changes significantly from the GTP (Figure 2G; from Kjeldgaard et al., 1993) to the GDP (Figure 2I; from Polekhina et al., 1996) states. It is clear that the architecture of EF-Tu within the kirromycin-stalled ternary complex (Figure 2H) resembles more closely the EF-Tu·GTP state in both, binary (Figure 2G) and ternary complexes (Figure 2C and D) than the GDP state (Figure 2I). This similarity with the GTP state is revealed by computation of cross-correlation coefficients between the crystal structures and the cryo-EM density (Table I). For E.coli, the X-ray structure was calculated in its GDP state (Song et al., 1999), and in order to obtain an acceptable fit, domain I needed to be moved significantly from its original position, close to domain III, to the position where the contacts between these two domains of the protein are minimized and enough room is created to accommodate the tRNA acceptor arm in the space available between them (Figure 3). Those changes produce a much better fit (Table I) and establish a GTP-state architecture. Figure 3.Docking of EF-Tu and aa-tRNA into the cryo-EM density of the ternary complex: (A) as seen from the solvent side; (B) as seen from the intersubunit side. (Views are similar, but not identical to those in Figure 2A and B.) Fitted atomic coordinates of E.coli EF-Tu (Song et al., 1999) and Phe-tRNAPhe [(from the ternary complex of T.aquaticus (Nissen et al., 1995)] are shown inside the semi-transparent cryo-EM density of the ternary complex. The domains of EF-Tu were fitted independently (domain I shown in green, domain II in yellow and domain III pink). The switch-I region within domain I is highlighted in cyan. The dotted line in (A) indicates the place of the kink in the anticodon arm of the tRNA. Orientations of the ribosome are shown as thumbnails on the left. SRL, α-sarcin–ricin loop; L11–rRNA (GAC), protein L11 and the segment of 58 nucleotides of the 23S rRNA, also known as the GTPase-associated center; S12, protein S12 from the small subunit; h5, helix 5 from the 16S rRNA; dc, decoding center in the A-site of the 30S subunit. Download figure Download PowerPoint Table 1. Correlation between the fitted X-ray structures and the cryo-EM density X-ray structure Rigid body fitting Flexible fitting T.aquaticus EF-Tu·GDP 0.51 – T.aquaticus EF-Tu·GTP 0.78 – E.coli EF-Tu·GDP 0.52 0.81 Phe-tRNAPhe·EF-Tu·GTP 0.69 0.83 Phe-tRNAPhe 0.77 0.81a a Value when the anticodon tip of the A-site tRNA was used for the fitting. In the case of the tRNA, the Phe-tRNAPhe within the ternary complex from T.aquaticus in the presence of a non-hydrolyzable GTP analog (Nissen et al., 1995) was used. It was not possible to get a full adjustment. The best docking was achieved by using the putative T and D loops in the elbow region of the L-shaped molecule as the main guide. The cryo-EM mass describes the above-mentioned change in the shape of the anticodon arm (‘kinked’ appearance; the position of the kink is indicated in Figure 3A by a dotted line), and this distortion causes the anticodon loop of the atomic coordinates of the Phe-tRNAPhe (Nissen et al., 1995) to lie partially outside the density mass (Figure 3A). Inside domain I of EF-Tu there is a segment of the sequence that overlaps with the fitted tRNA structure in the CCA end (Figure 3B). This part of the protein corresponds to a single turn of an α-helix known as the switch-I region (depicted in cyan in the figures), a putative ribosomal interaction site that undergoes a dramatic conversion in conformation to a β structure in the transition from the GTP to the GDP state (Abel et al., 1996). Obviously, either the position of this part of the protein or the position of the tRNA must be changed in order to avoid the overlap. Interaction of the ternary complex with the ribosome Fitting of X-ray structures into the cryo-EM map reveals three of the most conserved regions in the ribosome that are present in the immediate vicinity of the ternary complex: the decoding site on the 30S subunit (see Ogle et al., 2001); the L11–rRNA complex (Wimberly et al., 1999); and the SRL (Wool et al., 1992) at the base of the stalk of the large subunit. These ribosomal components were fitted in both subunits to study their interactions with Phe-tRNAPhe and with EF-Tu. The results of this analysis are shown in Figures 4 and 5. According to the positions found, some of the interactions can now be described. Figure 4.Interaction of EF-Tu and aa-tRNA with the ribosome. (A and B) Ribbons representation of the docked EF-Tu and aa-tRNA within the ternary complex. (C and D) Focus on the interaction between the α-sarcin–ricin loop (SRL) and the effector loop within domain I of EF-Tu (cyan). In (C) the coordinates of the whole ternary complex from T.aquaticus with a GTP analog (Nissen et al., 1995) were used for the fitting, while in (D) the crystal structure of EF-Tu from E.coli bound to GDP (Song et al., 1999) was used. Orientation of the ribosomes for (A) and (B) are shown as thumbnails on the left. Labeling is the same as in Figure 5. Download figure Download PowerPoint Figure 5.Interaction in the decoding center and accommodation of the aa-tRNA. (A–C) Semi-transparent representation of the ternary complex density from the cryo-EM map showing the fitted tRNA (gold) and the A-site tRNA (red) with corresponding mRNA codon (Yusupova et al., 2001). (C) A tRNA construct in which the anticodon position, up to the kink, is adopted from (B) and the rest of the tRNA from (A). (D–F) Interaction in the anticodon loop of the tRNA in the decoding site. H69, helix 69 from 23S rRNA; h44, helix 44 from 16S rRNA; h34, helix 34 from 16S rRNA; cd, A-site codon in the mRNA; AC, anticodon loop; S12, protein S12 in the 30S subunit. Download figure Download PowerPoint Turning first to the 50S subunit, the T loop of the aa-tRNA closely interacts with the region of the 58-nucleotide rRNA segment that binds to the L11 protein (Figure 4A and B). This portion of the ribosome is known as the GTPase-associated center (GAC) due to the fact that antibiotics that bind to it strongly affect the activity of EF-G and EF-Tu (Vazquez et al., 1979). The GAC is seen to interact with the T arm of the tRNA through a loop, around positions 1060–1075 in the 23S rRNA, which protrudes between the C- and the N-terminal domains of L11. In this loop, a single transversion in the residue 1067 impairs the function of the elongation factors (Saarma et al., 1997). Residues around position 2660 of the rRNA (within the SRL) were defined as binding site for EF-Tu by footprinting experiments (Moazed et al., 1988). The fitting leaves a distance between the SRL and EF-Tu large enough (10–15 Å) to be uncertain about the existence of a real binding contact. However, the effector loop region inside domain I of EF-Tu switches from an α-helical to a β-sheet structure and moves within the protein in the transition between GTP and GDP states (Abel et al., 1996). In Figure 4C and D, those two different positions for the effector loop are represented. When the coordinates of the EF-Tu·GTP form are used (Figure 4C), this switch-I region interacts with the SRL and avoids the earlier mentioned steric conflict with the CCA tip of the tRNA that the GDP state shows (Figure 4D). In the 30S subunit the ternary complex forms three contacts, one with domain II of EF-Tu and two with the aa-tRNA. Domain II contacts helix 5 of 16S rRNA; the acceptor arm of the tRNA interacts with the protein S12; and the anticodon loop reaches the decoding center. In Figure 4A and B it can be seen that protein S12 extends into a loop that goes close to the decoding center. This loop includes the conserved PNSA (Pro-Asn-Ser-Ala) amino acid sequence at positions 48–51. The mass in the region of the anticodon tip of the tRNA is unexpectedly wide, but the additional mass could be explained as the result of a conformational change of this loop of S12 such that it reaches the density in immediate vicinity to the tRNA (labeled with an asterisk in Figure 4A). Particular interest is focused on the codon–anticodon interaction and in the dynamic transition of the tRNA from the T- to the A-site. The rigid-body fitting of the tRNA structure inside the cryo-EM map leaves the anticodon loop partially outside the density (Figure 5A). Evidently, the tRNA crystal structure is different from the structure of the tRNA in the transition state observed here, and hence cannot be used to study the interactions in the region. However, it is important for understanding the process of tRNA accommodation that the anticodon portion of the tRNA interacting with the mRNA in the A-site (Agrawal et al., 2000a; Yusupova et al., 2001) partially overlaps the cryo-EM density of the tRNA up to the observed kink, and that the correlation coefficient is improved in the docking (Table I). The position of this A-site tRNA was deduced by the alignment of the P- and E-site tRNAs from the X-ray structure (Yusupova et al., 2001) with those from our density map. Figure 5C shows a merged representation of both tRNAs where the kink in the anticodon arm separates an A-site anticodon loop (red) from the rest of the tRNA still residing in the T-site (gold). We have used the accommodated position of the tRNA to represent the interactions in the decoding center. In Figure 5B the density identified with the anticodon is broadened on both sides. The extra mass not filled with the tRNA structure might be understood to be a result of contributions from the tight interactions of the anticodon end with the surrounding ribosomal elements (Figure 5D). This way, the parts of helix 69 from 23S rRNA and helix 44 from 16S rRNA, which interact tightly with the codon of the mRNA (Figure 5E), and as well as a putative contribution due to a conformational change of S12 (Figure 5F), would be displayed as a compact mass at the resolution of our map. Discussion Ternary complex in the presence of kirromycin The biggest problem that arose in our sample was its heterogeneity. Taking into account the occupancy of the ternary complex that the filter binding assays determined, it was reasonable to expect a significant fraction of the population of ribosomes to be
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