Study of the pattern of adverse events following immunization of children in a tertiary care hospital

Abstract

1Institut für Medizinische Physik und Biophysik, Charite—Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117 Berlin, Germany 2RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan 3AG Ribosomen, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany 4UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany 5Tohoku University Biomedical Engineering Research Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan 6Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S•EF-G ribosomal complex at 7.3 Å resolution and the crystal structure of EF-G-2•GTP, an EF-G homolog, at 2.2 Å resolution are presented. EF-G-2•GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation. Author Keywords: RNA The mechanism of EF-Tu appears to follow the classical scheme of G proteins ([Hilgenfeld, 1995] and [Yokosawa et al., 1973]) in that it is active in the GTP bound form and inactive in the GDP bound form: in the GTP form, EF-Tu has high affinity for aa-tRNAs, which is markedly decreased upon GTP hydrolysis. Local conformational changes in the GTP binding pocket of EF-Tu, which are in turn translated into a gross conformational change in the overall structure, are responsible for the change in affinity for aa-tRNA (Nyborg et al., 1996). Specifically, these local changes involve a restructuring of the switch I and II regions ([Abel et al., 1996] and [Polekhina et al., 1996]), which interact with the phosphate groups of the bound GTP and are named for the fact that they “switch” their conformations during the GTP-to-GDP transition. In contrast, the mechanism for EF-G-catalyzed translocation is not well understood and remains a controversial subject. Pre-steady-state studies ([Rodnina et al., 1997], [Savelsbergh et al., 2003] and [Wilden et al., 2006]) suggested that the binding of EF-G to the ribosome induces a conformational change leading to GTPase activation, after which EF-G exists in a GDP•Pi state in which the GTP binding pocket is closed and the inorganic phosphate (Pi) is not released. A subsequent rate-limiting step, “unlocking,” occurs, Pi is released (suggesting that the GTP binding pocket opens), and the tRNA-mRNA complex is moved. A modification to this pathway was presented by Pan and colleagues in which the conformational change associated with unlocking involves the movement of the P-tRNA into a P/E hybrid site while the A site tRNA moves afterwards (Pan et al., 2006). In an alternative model, Zavialov et al. proposed that the exchange of GDP for GTP on the ribosome promotes the “mRNA unlocking and translocation step,” which is followed by GTP hydrolysis (Zavialov et al., 2005). Regardless of the specific model used to describe translocation, all of the models propose that both the ribosome and EF-G undergo conformational changes that are important for GTPase activation. High-resolution X-ray structures of EF-G have not been forthcoming in revealing the structural nature of these conformational changes, as they have shown that the overall conformation of EF-G is essentially similar, regardless of the nature of the bound nucleotide ([Ævarsson et al., 1994], [al-Karadaghi et al., 1996], [Czworkowski et al., 1994], [Hansson et al., 2005b], [Hansson et al., 2005a] and [Laurberg et al., 2000]). Notably, in these structures, the electron density for the switch regions, which are important for coupling G-nucleotide binding to structural rearrangements, are either generally absent or display a large degree of disorder in the X-ray structures. The discontinuity between GTP/GDP binding and structural changes in EF-G is also evident in small angle X-ray scattering experiments, showing that the overall conformation of EF-G free in solution is similar, regardless of the nature of the bound nucleotide (Czworkowski and Moore, 1997). In contrast to the free EF-G structures, medium-resolution (11–18 Å) cryo-EM reconstructions revealed that, when bound to the ribosome, EF-G undergoes large-scale conformational changes ([Agrawal et al., 1999], [Frank and Agrawal, 2000], [Spahn et al., 2004], [Stark et al., 2000] and [Valle et al., 2003b]). In these models, a relative rearrangement in the five domains of EF-G results in domain IV being inserted into the A site and either pushing the A-tRNA into the P site and/or occupying the A site after translocation in order to prevent backward slippage of the tRNAs (Wilson and Noller, 1998). Cryo-EM also revealed that, upon EF-G binding, the ribosome undergoes a ratchet-like subunit rearrangement (RSR) in which the subunits twist relative to one another ([Spahn et al., 2001], [Spahn et al., 2004], [Frank and Agrawal, 2000] and [Valle et al., 2003b]). In particular, during the RSR, the head of the small ribosomal subunit rotates in the same direction as the tRNAs during translocation ([Schuwirth et al., 2005] and [Spahn et al., 2004]), potentially moving together to guide and control the translocation reaction. Since the X-ray structures suggested that nucleotide binding is not sufficient to switch the overall conformation of EF-G, a concerted effort between EF-G and the ribosome is likely to be involved in promoting these structural changes. Here we present a cryo-EM structure of the 70S•EF-G•GMPPNP complex from Thermus thermophilus at 7.3 Å resolution, as well as a 2.2 Å structure of T. thermophilus EF-G-2•GTP, a homolog of EF-G. The subnanometer resolution of the cryo-EM reconstruction allows the observation of the switch I region of EF-G and the characterization of its contacts within the ribosome•EF-G complex. The EF-G-2•GTP structure reveals conformational changes in EF-G-2 resulting from GTP binding. The overall conformation of EF-G-2•GTP, with the presence of an ordered switch I region, bears similarities to the cryo-EM reconstruction of EF-G•GMPPNP, indicating that EF-G-2•GTP has been crystallized in a conformation related to the active structure rather than the free solution structure represented by the previous EF-G structures. Collectively, the crystal structure and cryo-EM reconstruction provide a model to explain how coordinated communication between the ribosome and EF-G regulates the GTPase and translational activities of EF-G. For image processing, we recorded micrographs of a T. thermophilus 70S•EF-G complex in which EF-G was stalled on the ribosome, via the nonhydrolyzable GTP analog GMPPNP. Although the density for EF-G could be directly observed in the initial cryo-EM maps, the substoichiometric occupancy (see the Experimental Procedures) resulted in a fragmented appearance of EF-G, especially as the reconstruction progressed toward higher resolution. In order to solve this problem and to obtain a more homogeneous subset of particles, we employed a multireference 3D projection refinement procedure (Penczek et al., 2006) and selected a subset of particles bound by EF-G. Thus we selected 77,038 out of a total of 362,361 projections and obtained a final model (Figure 1) with a resolution of 7.3 Å, according to the FSC curve using the 0.5 cutoff criterion (see Figure S1 in the Supplemental Data available with this article online). In accordance with the resolution estimate, the grooves of the RNA helices are easily distinguished, the α-helical secondary structure within EF-G and the ribosomal proteins is observed, and in some cases the unstructured peptide chains corresponding to the extended tails of ribosomal proteins are resolved (Figure S2). In agreement with previous studies of the E. coli ribosome ([Spahn et al., 2001], [Frank and Agrawal, 2000] and [Valle et al., 2003b]), the T. thermophilus 70S•EF-G•GMPPNP complex undergoes RSR (Supplemental Results). Similar to X-ray studies of T. thermophilus ribosomes (Yusupov et al., 2001), our reconstruction also contains a tRNA that copurified with the 70S ribosomes (Figure 1; green density); however, here it is located in the P/E site rather than the E site (Supplemental Results). Therefore, biochemically, our complex is similar to those of previous studies, in which EF-G was trapped by a nonhydrolyzsable GTP analog on ribosomal complexes lacking an A-tRNA and containing a deacylated P-tRNA (Valle et al., 2003b). A few studies attempted to visualize a complex containing EF-G together with an aminoacylated A-tRNA (Agrawal et al., 1999), but it was later shown that the samples were heterogeneous, and the EF-G and tRNAs were present in different ribosome populations (Penczek et al., 2006). In half of the bacterial genomes sequenced to date, a gene for EF-G-2, an EF-G-like protein, has been identified. In the genome of T. thermophilus HB8, a 658 amino acid open reading frame named EF-G-2, with 34% identity and 56% similarity to EF-G (Figure S3), was identified. Overexpressed and purified EF-G-2 shows a detectable intrinsic GTPase activity in the absence of ribosomes (Figure 2A). In contrast, the ribosome-dependent GTPase assays demonstrate that EF-G-2 and EF-G are similarly activated by T. thermophilus ribosomes (Figure 2B). In in vitro poly(Phe) synthesis, EF-G-2 displays slightly lower activity than E