RelA/SpoT homolog (RSH) proteins play a central role in bacterial stringent response—a major stress-response pathway and key driver of bacterial virulence and antibiotic resistance (Neidhardt, 1987; Gentry et al., 2000; Pizarro-Cerda and Tedin, 2004; Dalebroux et al., 2010a; Gao et al., 2010; Nguyen et al., 2011; Dordel et al., 2014). In response to environmental stress, such as nutrient deprivation, RSH proteins synthesize small-molecule 'alarmones' collectively referred to as (p)ppGpp (i.e., guanosine pentaphosphate and guanosine tetraphosphate; [Cashel and Gallant, 1969] and reviewed in [Potrykus and Cashel, 2008a; Atkinson et al., 2011]). Accumulation of (p)ppGpp activates transcription of genes required for stress response, inhibits transcription of genes required for replication and growth, and reformats the transcription of metabolic genes according to the stress condition (Polakis et al., 1973; Mittenhuber, 2001; Magnusson et al., 2005; Jain et al., 2006a; Kuroda, 2006; Wang et al., 2007; Ferullo and Lovett, 2008; Potrykus and Cashel, 2008a; Traxler et al., 2008; Dalebroux et al., 2010a; Dalebroux et al., 2010b). Inactivation of RSH proteins in pathogenic bacteria dramatically reduces pathogenicity and bacterial load in the host, up to ~10,000-fold for Salmonella Typhimurium (Na et al., 2006; Sun et al., 2009; Dalebroux et al., 2010a; Vogt et al., 2011). Understanding the molecular mechanism of RSH activation may therefore guide the development of new antibacterial therapeutics.

In E. coli, RelA synthesizes alarmones in response to amino acid deprivation (Cashel and Gallant, 1969; Haseltine et al., 1972). When the supply of amino acids becomes limiting, binding of cognate but uncharged (deacylated) transfer RNA (tRNA) to the A (aminoacyl-tRNA) site of the 70S ribosome activates RelA (Haseltine and Block, 1973; Richter, 1976; Wendrich et al., 2002). The 70S•RelA•deacyl-tRNA complex triggers RelA to transfer a pyrophosphoryl group from ATP to GTP or to GDP to form pppGpp or ppGpp, respectively (Haseltine and Block, 1973; Sy and Lipmann, 1973; Wendrich et al., 2002). RelA also binds to the ribosome in the absence of deacyl-tRNA, but this binding does not stimulate (p)ppGpp synthesis (Haseltine and Block, 1973; Ramagopal and Davis, 1974; Wagner and Kurland, 1980; Wendrich et al., 2002).

RelA, a 744 amino-acid protein (~84 kDa), consists of functionally distinct halves. The N-terminal half (amino acids 1–380) includes a pseudo-hydrolase (inactive-hydrolase) domain (aa 1–200) and the (p)ppGpp synthetase domain (aa ~201–380). A crystal structure of the N-terminal half of the Streptococcus equisimilii RelA homolog RelSeq (aa 1–385) showed that the synthetase domain belongs to the nucleotidyltransferase superfamily and identified the catalytic residues (Hogg et al., 2004). The C-terminal half of RelA (aa ~400–744) and other RSH proteins is thought to control the synthetase activity of the N-terminal region (Schreiber et al., 1991; Gropp et al., 2001; Yang and Ishiguro, 2001a; Mechold et al., 2002; Avarbock et al., 2005; Jain et al., 2006b). Dimerization (Yang and Ishiguro, 2001a) or oligomerization (Gropp et al., 2001; Avarbock et al., 2005; Jain et al., 2006b) of free (ribosome-unbound) RelA and RSH proteins are thought to contribute to negative regulation of the synthetase activity. A 10.8-Å cryo-EM reconstruction of a 70S•RelA•deacyl-tRNA complex revealed a bi-lobed density overlapping with the elongation-factor-binding site near the A site (Agirrezabala et al., 2013). The position of RelA and conformation of deacyl-tRNA resembled those of elongation factor EF-Tu and aminoacyl tRNA in the 70S•EF-Tu•aa-tRNA pre-accommodation-like complexes (Stark et al., 2002; Valle, 2002; Schmeing et al., 2009). However, the resolution of the map did not allow model building, leaving the molecular details of RelA activation unclear (Agirrezabala et al., 2013). A lack of high-resolution structures of full-length RelA or its homologs bound to the ribosome precludes our ability to understand the regulation of RelA synthetase activity.

To understand how RelA activates stringent response on ribosomes bound with cognate deacyl-tRNA, we sought a high-resolution structure of the entire 70S•RelA•deacyl-tRNA complex. Single-particle electron cryo-microscopy (cryo-EM) and maximum-likelihood classification of a single dataset yielded four different cryo-EM structures of E. coli RelA bound to the E. coli ribosome at 3.9-Å to 4.1-Å resolution. As in cryo-EM structures of other ribosome complexes (e.g. [Greber et al., 2014; Fischer et al., 2015]), the local resolution of our maps in the ribosome core is higher than the average resolution of the maps, allowing for structural interpretation in central regions at near-atomic resolution. The structures reveal large-scale conformational rearrangements in RelA when it binds deacyl-tRNA entering the 30S A site, suggesting a mechanism of activation of the (p)ppGpp synthetase. Furthermore, distinct conformations of the deacyl-tRNA, 30S subunit, and the ribosomal decoding center reveal structural dynamics of tRNA binding in the presence of RelA and suggest why stringent response activation requires cognate tRNA.

We used single-particle cryo-EM to obtain the structures of full-length E. coli RelA bound to E. coli 70S•tRNA ribosome complex programmed with an mRNA coding for tRNA^fMet in the P site and tRNA^Phe in the A site. Maximum-likelihood classification using FREALIGN (Lyumkis et al., 2013) revealed four unique classes of ribosome particles containing RelA (Figure 1A–D, Figure 1—figure supplements 1 and 2, Figure 1—source data 1). In all RelA-bound structures, the ribosome contains P-site and E-site tRNAs and adopts the classical, non-rotated conformation (Cate et al., 1999; Frank and Agrawal, 2000; Yusupov et al., 2001), similar to that of the post-translocation-ribosome with peptidyl-tRNA in the P site (Voorhees et al., 2009; Jenner et al., 2010). In Structure I, the ribosome A site is vacant, and RelA is bound via its C-terminal portion while its N-terminal domains are disordered (Figure 1A). In Structures II, III, and IV, the central and C-terminal parts of RelA are well resolved and the anticodon-stem loop (ASL) of a cognate deacyl-tRNA^Phe is bound to the A site of the 30S subunit (Figure 1B–D). We refer to the deacyl-tRNA bound to the 30S A site and RelA as A/R (A/RelA) tRNA. Structures II, III, and IV differ from each other in the conformations of RelA, A/R tRNA, the 30S subunit, and the L11 stalk of the 50S subunit. The N-terminus of ribosomal protein L11 is required for RelA activation in 70S•RelA•deacyl-tRNA complexes (Friesen et al., 1974; Parker et al., 1976; Yang and Ishiguro, 2001b; Wendrich et al., 2002; Jenvert and Holmberg Schiavone, 2007; Shyp et al., 2012). The L11 N-terminus interacts with A/R tRNA but not with RelA in Structures II, III, and IV (Figure 1F–G, Figure 1—figure supplement 3). Thus, the lack of (p)ppGpp synthetase activity of RelA on mutant ribosomes missing L11 or the L11 N-terminus (Friesen et al., 1974; Wendrich et al., 2002; Jenvert and Holmberg Schiavone, 2007) is likely due to the inability of these ribosomes to coordinate deacyl-tRNA for activation of RelA.

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Figure 1—source data 1

Structure I-IV map resolution and refinement statistics.

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The C-terminal domain of RelA binds at intersubunit bridge B1a

Although activation of RelA catalysis requires cognate, deacylated A-site tRNA (Haseltine and Block, 1973), RelA can bind to the ribosomes in the absence of A-site tRNA (Ramagopal and Davis, 1974; Richter et al., 1975; Richter, 1976; Wendrich et al., 2002). Our map lacking A-site tRNA (Structure I) reveals density for the C-terminal region of RelA (aa ~530–744) near the A and P sites (Figure 1—figure supplement 2A). Specifically, the C-terminal region binds the intersubunit bridge B1a (Yusupov et al., 2001), which connects the A-site finger (helix 38) of the large subunit to the head of the small subunit (Figures 1E–G and 2A). The lack of density for the central and N-terminal domains of RelA suggests that these regions are not ordered in the absence of deacylated A-site tRNA (Figure 1—figure supplement 2A).

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The resolved C-terminal region comprises two domains. The ACT domain (aspartate kinase-chorismate mutase-tyrA; residues 665–744; [Atkinson et al., 2011]) is composed of four β-strands covered by two α-helices (Grant, 2006), and lies in the 50S subunit within a cavity formed by the A-site finger, helix 89, L16 and P-site tRNA (Figure 2A–B and Figure 2—figure supplement 1). The tips of two β-hairpins, including Q705-Q706 and highly conserved D675, interact with R50 and R51 of L16 (Figure 2B). The β-strands of ACT form a platform that packs against the 3´-strand of the A-site finger. Here, the N-terminal and the C-terminal β-strands of ACT, including R670 and R739, interact with the bulged A896 of the A-site finger helix (Figure 2B and Figure 2—figure supplement 1A). The fold of the ACT domain was previously noted to resemble the RNP motif that binds single-stranded RNA, but no RNA-binding ACT domains had been identified (Burd and Dreyfuss, 1994; Grant, 2006). Our findings reveal that while the RelA ACT domain is not sequence-homologous to RNP motifs, it interacts with double-stranded RNA via the same face that RNP domains use to bind single-stranded RNA.

The second binding interface between the C-terminal region and the ribosome involves a previously unclassified RelA domain (aa 585–660). The domain bridges the large and the small ribosomal subunits (Figures 1E–F, 2A–B and Figure 2—figure supplement 1B). We refer to this domain as RIS (Ribosome-InterSubunit) domain. The domain core contains a four-stranded β-sheet and a short α-helix and is structurally similar to a zinc-finger domain (Lee et al., 1989). On the 50S subunit, the α-helix (residues 638–647) docks into the minor groove of the A-site finger and interacts with 23S rRNA residues 883–885 and 892–894 (Figure 2A–B). On the 30S subunit, the β-sheet of RIS packs at the hydrophobic patch of the β-sheet of S19, comprising V57, P58 and F60.

In summary, Structure I reveals that the ACT and RIS domains of RelA form a C-terminal superdomain that anchors RelA to the 70S ribosome, consistent with reduced binding of RelA to ribosomes upon mutation or deletion of the C-terminal domain (Yang and Ishiguro, 2001a). The density for the RIS domain core is well resolved in Structure I (Figure 1—figure supplement 2A), supporting the model that amino acids encompassing the RIS domain form the major ribosomal-binding domain of RelA (Yang and Ishiguro, 2001a).

Deacyl-tRNA pins the TGS domain against the 30S subunit, exposing the dynamic N-terminal domains near the spur

Structures II, III, and IV contain RelA bound to the ribosome with cognate deacyl-tRNA in the A site of the 30S subunit (Figure 1B–D). The ribosome structures share an overall conformation, including the relative positions of ribosomal subunits, tRNAs and RelA. As described in the earlier cryo-EM study of RelA ribosome complexes (Agirrezabala et al., 2013), the positions of A/R tRNA in the RelA-bound structures globally resemble that of the A/T pre-accommodated aminoacyl-tRNA captured in the ribosome in the presence of EF-Tu (Stark et al., 2002; Valle, 2002; Schmeing et al., 2009). However, we observe important differences between the A/R and A/T tRNAs, and among ribosome conformations, as discussed in a following section.

Structures II, III, and IV reveal density for the entire RelA protein when cognate deacyl-tRNA is bound in the 30S A site. In these structures, RelA wraps around the tRNA and adopts a boomerang-like conformation (Figure 1F). This RelA conformation contrasts with the compact RelA conformation occupying the elongation-factor binding site that was proposed based on the 10.8-Å map of a 70S•RelA•deacyl-tRNA complex (Agirrezabala et al., 2013). However, our re-evaluation of the lower-resolution map revealed previously unassigned density near the intersubunit bridge B1a, which makes that map consistent with the extended RelA conformation shown here (Figure 2—figure supplement 2).

In Structures II, III and IV, the C-terminal RIS and ACT domains form one wing of the boomerang-like structure at bridge B1a and are positioned similarly to those in Structure I (Figure 2A–B). The N-terminal part of the RIS domain (at aa 590–595) is also stabilized by interactions with the phosphate backbone of G987 and the 954–960 loop of the 16S ribosomal RNA (rRNA), which forms a wall of the A-site tRNA-binding cavity. The C-terminal domains are connected with the central α-helical domain (AH; aa 485–580; Figure 2B–C). The core of the AH domain (aa ~520–560) is formed by short helices, which pack near the D stem of the A/R tRNA. The ~15-amino-acid C-terminal helix of the AH domain connects the AH core with the RIS domain (Linker helix 2; Figure 2B–C). Linker helix 2 packs against the ACT domain similarly to an α-helix of the dimerization partner of the isolated ACT domain of Chlorobium tepidum RSH (PDB: 2KO1; [Eletsky et al., 2009]) (Figure 2D). This suggests that if the ACT domains were also dimerized in the full-length free RelA, the packing of this α-helical linker next to the ACT domain would be possible on the ribosome only upon dimer disassembly. The long N-terminal helix (Linker Helix 1), which lies under the acceptor arm of the A/R tRNA, connects the AH core with the ubiquitin-like TGS domain (amino acids 405–470; ThrRS, GTPase, and SpoT/RelA (Sankaranarayanan et al., 1999) (Figure 2C). Together the central AH and TGS domains form the elbow of the RelA boomerang. The TGS domain is pinned against 16S rRNA by the acceptor end of A/R tRNA (Figure 3A–B), as described below. The overall conformations of the TGS domain are similar between Structures II, III, and IV (Figure 3—figure supplement 1A). Finally, the N-terminal pseudo-hydrolase domain (aa 1–200) and synthetase domain (aa 200–380), forming the second wing of the boomerang, face the periphery of the ribosome in the vicinity of the 30S subunit spur (helix 6) (Figure 4A) and adopt a range of conformations in Structures II, III and IV, as discussed below.

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The TGS domain interacts with the 3´ CCA end of deacylated A/R tRNA and with 16S rRNA helix 5 (Figure 3A). The ⁷⁴CCA⁷⁶ end adopts a conformation similar to that in the EF-Tu-bound A/T-tRNA, in which C75 is bulged out (Schmeing et al., 2009; Fischer et al., 2015), whereas C74 and A76 interact with several conserved residues of the protein. H432 stabilizes the CCA conformation by intercalating between C74 and C75 (Figure 3B). P411 and K412 on a β-hairpin loop interact with the nucleobase of A76 (Figure 3B). The ribose of A76 faces a β-sheet at residues 461–465 (Figure 3B). (p)ppGpp synthesis by RelA requires deacyl-tRNA binding to the ribosome (Haseltine and Block, 1973). An amino acid bound at A76 would sterically clash with the β-sheet of TGS and prevent binding of A/R tRNA to the TGS domain, in keeping with the inability of charged tRNA to activate RelA. To gain insights into the role of interaction between the CCA end and the TGS domain, we compared our structures with the X-ray structure of the isolated TGS domain from C. leptum RSH (PDB: 3HVZ) (Forouhar et al., 2009). The isolated TGS domain forms a dimer. The comparison reveals that the A/R tRNA disrupts the dimerization interface (Figure 3C). Since oligomerization of RSH proteins inhibits (p)ppGpp production (Gropp et al., 2001; Yang and Ishiguro, 2001a; Avarbock et al., 2005; Jain et al., 2006b), interaction with deacyl-tRNA may contribute to RelA activation via stabilization of the monomeric RelA.

The N-terminal pseudo-hydrolase and synthetase domains in the intersubunit space of the ribosome were poorly resolved in the maps for Structures II, III and IV, suggesting structural heterogeneity. To resolve this heterogeneity, we performed data sub-classification, using a focused spherical mask (Grigorieff, 2016) covering the N-terminal region of RelA, separately for Structures II, III and IV. Sub-classification of each structure into three or more classes revealed two predominant classes that resolved the shapes of the N-terminal region, which differ in position by at least 10 Å (Figure 4—figure supplement 1A–F). Classification into seven classes reveals the appearance of less resolved density between these two extreme conformations, suggesting that the N-terminal domain samples a continuum of positions (Figure 4—figure supplement 1G). The maps of the two predominant conformations allowed rigid-body fitting of the homology model of the E. coli RelA pseudo-hydrolase and synthetase domains, obtained from the crystal structure of the isolated N-terminal domain from S. equisimilis RelSeq (Hogg et al., 2004). In the first conformation, the synthetase domain is positioned near the spur. The synthetase domain contains a long helix (the spine helix, at aa 208–243), which buttresses the catalytic region at the β-platform (aa 248–340; Figure 4A and C) (Hogg et al., 2004). The loop, which connects the spine helix with the β-platform (at residues 244–246) approaches the tip of the 16S spur within ~5 Å, suggesting that the protein interacts with rRNA, but the details of the possible interaction cannot be visualized in the low-resolution maps (Figure 4—figure supplement 1A–C). In the second conformation, the synthetase domain is separated from the spur by shifting away from its first predominant conformation by ~10 Å (Figure 4B). The pseudo-hydrolase domain is bound near the sarcin-ricin loop of the large subunit (nt 2653–2667 of 23S rRNA). The homologous RelSeq contains a functional hydrolase domain, and allosteric regulation was proposed to govern the switch between (p)ppGpp hydrolase and synthetase activities (Hogg et al., 2004). It is possible that activation of the synthetase domain of RelA involves conformational rearrangements between the pseudo-hydrolase and synthetase domains, triggered by movement relative to the TGS domain and/or interactions with ribosomal RNA. Alternatively, specific interactions of the synthetase domain with the spur may trigger the catalytic activity. The RelA synthetase domain structure resembles that of other second-messenger synthetases (Figures 4D and Figure 4—figure supplement 2), including: metazoan innate immune sensor OAS1, a 2′–5′–oligoadenylate synthase triggered by double-stranded RNA (Donovan et al., 2013; Lohöfener et al., 2015); metazoan cGAS, a cyclic-GMP–2′–5′–AMP synthase triggered by double-stranded DNA (Civril et al., 2013; Gao et al., 2013; Kranzusch et al., 2013; Sun et al., 2013); and Vibrio cholerae pathogenicity factor DncV, a cyclic-GMP–3′–5′–AMP synthase (Kranzusch et al., 2014). Innate immune sensors OAS1 and cGAS are activated by binding of an RNA or DNA duplex, respectively, at the spine helix (Civril et al., 2013; Donovan et al., 2013; Gao et al., 2013; Lohöfener et al., 2015). The proximity of the spine helix of RelA to the spur highlights the possibility of activation of RelA via a mechanism reminiscent of those for OAS1 and cGAS innate immune sensors (Figure 4—figure supplement 2).

Distinct intermediates of tRNA in the 30S A site of Structures II, III, and IV

Our structures reveal three conformations of the deacyl-tRNA and the 30S decoding center, providing insights into the mechanism of specific RelA activation by cognate tRNA (shown as an animation in Video 1 and http://labs.umassmed.edu/korostelevlab/msc/relamovie.gif). Activation of (p)ppGpp synthesis by RelA on the ribosome depends on the selection of tRNA cognate to the mRNA codon in the A site (Haseltine and Block, 1973). In their in vitro experiments, Haseltine and Block demonstrated that the substitution of cognate tRNA^Ala, which reads the GCA, GCC, GCG or GCU codons, with near-cognate tRNA^Val (GUA, GUC, GUG, or GUU) or tRNA^Glu (GAA or GAG) results in a more than 32-fold decrease in (p)ppGpp synthesis by RelA on ribosomes programmed with an Ala codon in the A site (Haseltine and Block, 1973). Our structures suggest stepwise accommodation of the codon-anticodon helix in the decoding center, which helps explain this exquisite sensitivity.

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Classification of our cryo-EM data revealed three unique classes (Structures II, III, and IV) that differ in the conformation of A/R tRNA (Figures 1B–D and Figure 1—figure supplement 1). In all three structures, the A/R tRNA anticodon base-pairs with the mRNA codon in the 30S A site, the elbow contacts the L11 stalk, whereas the acceptor arm is located in the vicinity of the sarcin-ricin loop of 23S rRNA (Figure 1F–G). The A/R tRNAs are highly distorted relative to accommodated A-site tRNA, such that the anticodon-stem loop is kinked toward the CCA end of the tRNA (Figure 5—figure supplement 1A), somewhat similar to the A/T aminoacyl-tRNA in EF-Tu-bound pre-accommodation-like ribosome structures (Stark et al., 2002; Valle, 2002; Schmeing et al., 2009; Fischer et al., 2015). The A/R tRNAs, however, differ from the A/T tRNA in the degrees of twisting around residues 26 and 44, which link the anticodon-stem loop with the rest of tRNA (Figure 5—figure supplement 1B). The CCA end of A/R tRNA (Structure II) is positioned ~10 Å away from that in the A/T tRNA (Figure 5—figure supplement 1B). Relative to the P-site tRNA, the A/R elbows of all three RelA-bound structures are tilted by up to 10 Å farther than the A/T tRNA (Figure 5—figure supplement 1A). As such, the A/R tRNA appears even more slanted away from the ribosome core than the A/T tRNA.

Comparison of Structures II, III, and IV reveals a concerted movement of A/R tRNA and the RelA central domains toward the head of the 30S subunit, as if the tRNA gradually 'settles' into the A site of the 30S subunit from Structure II through III to IV (Figure 5A–B). The tRNA accommodation coincides with a conformational change in the 30S subunit termed 'domain closure' (Ogle et al., 2001; Ogle et al., 2002; Jenner et al., 2010; Demeshkina et al., 2012). The acceptor arm of A/R tRNA and the TGS domain of RelA shift toward the head of the 30S subunit by ~2 Å between Structures II and III, and by ~2 Å between Structures III and IV (Figure 5C–D and Figure 6—figure supplement 1). The shoulder of the 30S subunit also moves by nearly 5 Å toward the head and body from Structure II to IV (Figure 6—figure supplement 1 and Figure 6—source data 1). In Structure II, the 30S subunit is in the open conformation observed previously in the absence of A-site tRNA (Ogle et al., 2001; Jenner et al., 2010), whereas in Structure IV, the 30S subunit is in the fully closed conformation (Figure 6A–B and Figure 6—figure supplement 1) (Selmer et al., 2006; Jenner et al., 2010; Demeshkina et al., 2012). The 30S subunit in Structure III adopts an intermediate state between the open and closed states (Figures 6B and Figure 6—figure supplement 1). Thus, Structure II represents a previously unseen open 30S subunit in the presence of the codon-anticodon interaction. Structure III, in turn, represents an intermediate in the 30S 'domain-closure' pathway.

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Distances between Structures II, III and IV, reflecting the movement of the 30S shoulder domain from Structures II to III to IV, relative to the head and the body of the 30S subunit.

https://doi.org/10.7554/eLife.17029.020

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The observation of open, intermediate, and closed conformations of the 30S subunit with A-site tRNA interacting with the mRNA codon prompted us to study the conformation of the decoding center in each structure in more detail. The local resolution of our maps in the decoding center is sufficient to determine nucleotide conformations (Figure 6—figure supplements 2 and 3). Studies of ribosome-tRNA complexes demonstrated that the decoding center plays a central role in cognate tRNA stabilization. Specifically, universally conserved nucleotides of the decoding center A1492, A1493, G530 of 16S rRNA and A1913 of 23S rRNA interact with the minor groove of the codon-anticodon interaction (Ogle et al., 2001; Selmer et al., 2006; Jenner et al., 2010). A1492 and A1493 are important in stabilizing Watson-Crick geometry of the first two base pairs (Ogle et al., 2001; Demeshkina et al., 2012), and are thus thought to contribute to tRNA recognition, providing high fidelity of protein synthesis (Ogle et al., 2003).

Despite the presence of an A-site tRNA in Structure II, the conformation of the decoding center resembles that found in the absence of an A-site tRNA with the 30S in an open conformation (compare Figure 6E–D) (Ogle et al., 2001; Jenner et al., 2010). Strong density shows that A1492 resides inside helix 44, as observed in the absence of an A-site tRNA (A1492 OFF) (Ogle et al., 2001; Jenner et al., 2010) (Figures 6E and Figure 6—figure supplement 3B,F and I). G530 is separated from A1492 by more than 10 Å (Figure 6E and Figure 6—figure supplement 3B), similar to that in the absence of an A-site tRNA (G530 in the OFF position). A1493 bulges out from helix 44 of 16S rRNA, so that the nucleotide is oriented toward the codon-anticodon helix. Weak density suggests that the base does not form a stable interaction with the codon-anticodon helix (Figure 6—figure supplement 3I), although A1493 appears pre-arranged for such an interaction by being bulged out (A1493 in the ON position). In summary, the key decoding center nucleotides in Structure II adopt the following conformations: A1493 ON, A1492 OFF, and G530 OFF.

In the decoding center of Structure III — corresponding to the intermediate state of domain closure — A1493 and A1492 contact the codon-anticodon helix, forming A-minor interactions with the first and second codon-anticodon base pairs (i.e., A1493 ON and A1492 ON; Figure 6F). G530 is in the anti conformation and shifted, along with the shoulder of the 30S subunit, toward A1492 (Figures 6C,F and Figure 6—figure supplement 3C,G,J). Here, G530 adopts a position between that in ribosomes with 'vacant' and 'filled' A sites (G530 SEMI-ON). The decoding center nucleotides in Structure III therefore adopt the conformations A1493 ON, A1492 ON, and G530 SEMI-ON.

Finally, the decoding-center nucleotides in Structure IV adopt conformations nearly identical (Figures 6G and Figure 6—figure supplement 3D, H, K) to those in the A-tRNA-bound ribosome (A1493/A1492/G530 ON) (Selmer et al., 2006; Jenner et al., 2010; Demeshkina et al., 2012). The 530 loop is shifted closer to A1493 and A1492, so that G530 interacts with A1492. This shift is coupled with the 30S subunit closure, also observed in 70S complexes with A-site tRNA (Selmer et al., 2006; Jenner et al., 2010; Demeshkina et al., 2012) and pre-accommodation-like 70S•EF-Tu•aa-tRNA complexes (Stark et al., 2002; Valle, 2002; Schmeing et al., 2009; Fischer et al., 2015).

Structural mechanism of tRNA decoding in the A site

Our observation of the open and intermediate states at the decoding center in Structures II and III suggests how cognate tRNA is specifically selected during RelA activation. We propose the following structural mechanism of deacyl-tRNA decoding (Video 1 and Figure 7). At early steps, interaction of the anticodon stem loop of a cognate or non-cognate tRNA occurs with the domain-open conformation of the 30S subunit, in which the decoding nucleotides are not positioned to stabilize the codon-anticodon helix. At this stage, the non-cognate tRNA dissociates prior to the closure of the 30S subunit, as the latter would require formation of the A-minor interactions by A1492 and A1493 with the Watson-Crick-paired codon-anticodon helix (Ogle et al., 2001; Jenner et al., 2010). In the case of cognate tRNA, acceptance of tRNA and 30S domain closure would be coupled with the formation of the A1492-G530 bridge (i.e., G530 ON) to stabilize the tRNA on the ribosome. The A1493-ON and/or A1493/A1492-ON states in Structures II and III may therefore serve as checkpoints on the path of acceptance of Watson-Crick base-paired tRNA and mRNA.

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Our observation of previously elusive states of tRNA binding also suggests insights into the accuracy of aminoacyl-tRNA selection during elongation, a universally conserved mechanism responsible for the accurate transfer of genetic information. Despite a wealth of structural, biochemical and biophysical studies, the high-resolution structural understanding of this mechanism is limited and distinct mechanistic models have been proposed (e.g. [Ogle et al., 2001; Demeshkina et al., 2012]). Current structural understanding is limited to observation of the open and the closed conformations of the 30S subunit in the absence and presence of A-site tRNA, respectively (Ogle et al., 2001; Jenner et al., 2010). Biochemical (Rodnina et al., 1994; Rodnina et al., 1995; Rodnina et al., 1996; Pape et al., 1998) and biophysical (Blanchard et al., 2004; Gonzalez et al., 2007) studies demonstrated that tRNA accommodation includes an early short-lived intermediate – prior to forming the A/T state – during which non-cognate tRNAs can be rejected. However, early or intermediate states prior to the formation of the A/T state have not been structurally visualized at high resolution. Our observation of incompletely engaged tRNA in the domain-open or intermediate state of the 30S subunit suggests that similar states exist for the aminoacyl-tRNA•EF-Tu•GTP ternary complex. The tRNA acceptor arm positions are separated by ~5 Å in the early pre-accommodation step (Structure II) and the domain-closed step tRNA (Structure IV). This could be sufficient to keep EF-Tu farther from the GTPase-activating sarcin-ricin loop of 23S rRNA (Voorhees et al., 2010) at an early step, allowing non-cognate ternary complex dissociation prior to GTP hydrolysis, EF-Tu release and tRNA acceptance via domain closure. Further structural and biochemical studies of the pre-accommodating aminoacyl-tRNA•EF-Tu•GTP ternary complexes are required to test whether tRNA binding during the stringent response and during protein elongation are structurally similar.

1. 1. Loveland AB
   2. Bah E
   3. Madireddy R
   4. Zhang Y
   5. Brilot AF
   6. Grigorieff N
   7. Korostelev AA

   (2016) Structure of RelA bound to ribosome in absence of A/R tRNA (Structure I)

   Publicly available at the RCSB Protein Data Bank (accession no: 5KPS).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5KPS
2. 1. Loveland AB
   2. Bah E
   3. Madireddy R
   4. Zhang Y
   5. Brilot AF
   6. Grigorieff N
   7. Korostelev AA

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Author details

1. Anna B Loveland

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
   3. Department of Biochemistry, Brandeis University, Waltham, United States
   4. Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States

   Contribution

   ABL, Wrote the manuscript, Collected and analyzed cryo-EM data, Contributed to manuscript finalization, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

2. Eugene Bah

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Present address

   Mayo Medical School, Rochester, United States

   Contribution

   EB, Prepared 70S•RelA complexes, Acquisition of data, Contributed unpublished essential data or reagents, Contributed to manuscript finalization

   Competing interests

   No competing interests declared.

3. Rohini Madireddy

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   RM, Subcloned the relA gene and assisted in 70S•RelA complex preparation, Contributed unpublished essential data or reagents, Contributed to manuscript finalization

   Competing interests

   No competing interests declared.

4. Ying Zhang

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   YZ, Prepared 70S•RelA complexes, Contributed unpublished essential data or reagents, Contributed to manuscript finalization

   Competing interests

   No competing interests declared.

5. Axel F Brilot

   1. Department of Biochemistry, Brandeis University, Waltham, United States
   2. Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States

   Present address

   1. Department of Biochemistry and Biophysics, University of California, San Francisco, United States
   2. School of Medicine, University of California, San Francisco, United States

   Contribution

   AFB, Assisted with cryo-EM data collection and analyses, Contributed to manuscript finalization

   Competing interests

   No competing interests declared.

6. Nikolaus Grigorieff

   1. Department of Biochemistry, Brandeis University, Waltham, United States
   2. Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States
   3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   NG, Oversaw cryo-EM data processing, Designed the project, Wrote the manuscript, Analysis and interpretation of data, Contributed to manuscript finalization

   For correspondence

   niko@grigorieff.org

   Competing interests

   NG: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X

7. Andrei A Korostelev

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   AAK, Built and refined structural models, Wrote the manuscript, Designed the project, Analysis and interpretation of data, Contributed to manuscript finalization

   For correspondence

   andrei.korostelev@umassmed.edu

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X

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