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.

Figure 1 with 4 supplements see all

Download asset Open asset

Figure 1—source data 1

Structure I-IV map resolution and refinement statistics.

https://doi.org/10.7554/eLife.17029.003

Download elife-17029-fig1-data1-v2.doc

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).

Figure 2 with 2 supplements see all

Download asset Open asset

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.

Figure 3 with 1 supplement see all

Download asset Open asset

Figure 4 with 2 supplements see all

Download asset Open asset

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.

Video 1

Download asset

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.

Figure 5 with 1 supplement see all

Download asset Open asset

Figure 6 with 3 supplements see all

Download asset Open asset

Figure 6—source data 1

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

Download elife-17029-fig6-data1-v2.doc

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.

Figure 7 with 1 supplement see all

Download asset Open asset

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

   (2016) Structure of RelA bound to ribosome in presence of A/R tRNA (Structure II)

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

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5KPV
3. 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 presence of A/R tRNA (Structure III)

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

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5KPW
4. 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 presence of A/R tRNA (Structure IV)

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

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

   (2016) RelA bound to 70S ribosome in absence of A/R tRNA (Structure I)

   Publicly available at the EMDataBank (accession no: 8279).

   http://emsearch.rutgers.edu/atlas/8279_summary.html
6. 1. Loveland AB
   2. Bah E
   3. Madireddy R
   4. Zhang Y
   5. Brilot AF
   6. Grigorieff N
   7. Korostelev AA

   (2016) RelA bound to 70S ribosome in presence of A/R tRNA (Structure II)

   Publicly available at the EMDataBank (accession no: 8280).

   http://emsearch.rutgers.edu/atlas/8280_summary.html
7. 1. Loveland AB
   2. Bah E
   3. Madireddy R
   4. Zhang Y
   5. Brilot AF
   6. Grigorieff N
   7. Korostelev AA

   (2016) RelA bound to 70S ribosome in presence of A/R tRNA (Structure III)

   Publicly available at the EMDataBank (accession no: 8281).

   http://emsearch.rutgers.edu/atlas/8281_summary.html
8. 1. Loveland AB
   2. Bah E
   3. Madireddy R
   4. Zhang Y
   5. Brilot AF
   6. Grigorieff N
   7. Korostelev AA

   (2016) RelA bound to 70S ribosome in presence of A/R tRNA (Structure IV)

   Publicly available at the EMDataBank (accession no: 8282).

   http://emsearch.rutgers.edu/atlas/8282_summary.html

1. 1. Agirrezabala X
   2. Fernández IS
   3. Kelley AC
   4. Cartón DG
   5. Ramakrishnan V
   6. Valle M

   (2013) The ribosome triggers the stringent response by RelA via a highly distorted tRNA

   EMBO Reports 14:811–816.

   https://doi.org/10.1038/embor.2013.106

   • Google Scholar
2. 1. Arenz S
   2. Abdelshahid M
   3. Sohmen D
   4. Payoe R
   5. Starosta AL
   6. Berninghausen O
   7. Hauryliuk V
   8. Beckmann R
   9. Wilson DN

   (2016) The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis

   Nucleic Acids Research 44:6471–6481.

   https://doi.org/10.1093/nar/gkw470

   • Google Scholar
3. 1. Atkinson GC
   2. Tenson T
   3. Hauryliuk V

   (2011) The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life

   PLoS One 6:e23479.

   https://doi.org/10.1371/journal.pone.0023479

   • Google Scholar
4. 1. Avarbock A
   2. Avarbock D
   3. Teh JS
   4. Buckstein M
   5. Wang ZM
   6. Rubin H

   (2005) Functional regulation of the opposing (p)ppGpp synthetase/hydrolase activities of RelMtb from Mycobacterium tuberculosis

   Biochemistry 44:9913–9923.

   https://doi.org/10.1021/bi0505316

   • Google Scholar
5. 1. Bairoch A
   2. Boeckmann B
   3. Ferro S
   4. Gasteiger E

   (2004) Swiss-Prot: juggling between evolution and stability

   Briefings in Bioinformatics 5:39–55.

   https://doi.org/10.1093/bib/5.1.39

   • Google Scholar
6. 1. Blanchard SC
   2. Gonzalez RL
   3. Kim HD
   4. Chu S
   5. Puglisi JD

   (2004) tRNA selection and kinetic proofreading in translation

   Nature Structural & Molecular Biology 11:1008–1014.

   https://doi.org/10.1038/nsmb831

   • Google Scholar
7. 1. Brilot AF
   2. Korostelev AA
   3. Ermolenko DN
   4. Grigorieff N

   (2013) Structure of the ribosome with elongation factor G trapped in the pretranslocation state

   PNAS 110:20994–20999.

   https://doi.org/10.1073/pnas.1311423110

   • Google Scholar
8. 1. Brown A
   2. Fernández IS
   3. Gordiyenko Y
   4. Ramakrishnan V

   (2016) Ribosome-dependent activation of stringent control

   Nature 534:277–280.

   https://doi.org/10.1038/nature17675

   • Google Scholar
9. 1. Burd CG
   2. Dreyfuss G

   (1994) Conserved structures and diversity of functions of RNA-binding proteins

   Science 265:615–621.

   https://doi.org/10.1126/science.8036511

   • Google Scholar
10. 1. Cashel M
    2. Gallant J

    (1969) Two compounds implicated in the function of the RC gene of Escherichia coli

    Nature 221:838–841.

    https://doi.org/10.1038/221838a0

    • Google Scholar
11. 1. Cate JH
    2. Yusupov MM
    3. Yusupova GZ
    4. Earnest TN
    5. Noller HF

    (1999) X-ray crystal structures of 70S ribosome functional complexes

    Science 285:2095–2104.

    https://doi.org/10.1126/science.285.5436.2095

    • Google Scholar
12. 1. Chapman MS

    (1995) Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function

    Acta Crystallographica Section a Foundations of Crystallography 51:69–80.

    https://doi.org/10.1107/S0108767394007130

    • Google Scholar
13. 1. Chen JZ
    2. Grigorieff N

    (2007) SIGNATURE: a single-particle selection system for molecular electron microscopy

    Journal of Structural Biology 157:168–173.

    https://doi.org/10.1016/j.jsb.2006.06.001

    • Google Scholar
14. 1. Civril F
    2. Deimling T
    3. de Oliveira Mann CC
    4. Ablasser A
    5. Moldt M
    6. Witte G
    7. Hornung V
    8. Hopfner KP

    (2013) Structural mechanism of cytosolic DNA sensing by cGAS

    Nature 498:332–337.

    https://doi.org/10.1038/nature12305

    • Google Scholar
15. 1. Cornish PV
    2. Ermolenko DN
    3. Noller HF
    4. Ha T

    (2008) Spontaneous intersubunit rotation in single ribosomes

    Molecular Cell 30:578–588.

    https://doi.org/10.1016/j.molcel.2008.05.004

    • Google Scholar
16. 1. Dalebroux ZD
    2. Svensson SL
    3. Gaynor EC
    4. Swanson MS

    (2010a) ppGpp conjures bacterial virulence

    Microbiology and Molecular Biology Reviews 74:171–199.

    https://doi.org/10.1128/MMBR.00046-09

    • Google Scholar
17. 1. Dalebroux ZD
    2. Yagi BF
    3. Sahr T
    4. Buchrieser C
    5. Swanson MS

    (2010b) Distinct roles of ppGpp and DksA in legionella pneumophila differentiation

    Molecular Microbiology 76:200–219.

    https://doi.org/10.1111/j.1365-2958.2010.07094.x

    • Google Scholar
18. Software

    1. DeLano WL

    (2002)

    The pymOL molecular graphics system

    Delano Scientific, Palo Alto, CA.
19. 1. Demeshkina N
    2. Jenner L
    3. Westhof E
    4. Yusupov M
    5. Yusupova G

    (2012) A new understanding of the decoding principle on the ribosome

    Nature 484:256–259.

    https://doi.org/10.1038/nature10913

    • Google Scholar
20. 1. Donovan J
    2. Dufner M
    3. Korennykh A

    (2013) Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1

    PNAS 110:1652–1657.

    https://doi.org/10.1073/pnas.1218528110

    • Google Scholar
21. 1. Dordel J
    2. Kim C
    3. Chung M
    4. Pardos de la Gándara M
    5. Holden MT
    6. Parkhill J
    7. de Lencastre H
    8. Bentley SD
    9. Tomasz A

    (2014) Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus

    mBio 5:e01000.

    https://doi.org/10.1128/mBio.01000-13

    • Google Scholar
22. Data

    1. Eletsky A
    2. Garcia E
    3. Wang H
    4. Ciccosanti C
    5. Jiang M
    6. Nair R
    7. Rost B
    8. Acton TB
    9. Xiao R
    10. Everett JK
    11. Lee H
    12. Prestegard JH
    13. Montelione GT
    14. Szyperski T
    15. Northeast Structural Genomics Consortium

    (authors) (2009)

    Solution NMR structure of the ACT domain from GTP pyrophosphokinase of chlorobium tepidum. Northeast Structural Genomics Consortium Target CtR148A

    RCSB Protein Data Bank.
23. 1. Elf J
    2. Ehrenberg M

    (2005) Near-critical behavior of aminoacyl-tRNA pools in E. coli at rate-limiting supply of amino acids

    Biophysical Journal 88:132–146.

    https://doi.org/10.1529/biophysj.104.051383

    • Google Scholar
24. 1. Engh RA
    2. Huber R

    (1991) Accurate bond and angle parameters for X-ray protein structure refinement

    Acta Crystallographica Section a Foundations of Crystallography 47:392–400.

    https://doi.org/10.1107/S0108767391001071

    • Google Scholar
25. 1. English BP
    2. Hauryliuk V
    3. Sanamrad A
    4. Tankov S
    5. Dekker NH
    6. Elf J

    (2011) Single-molecule investigations of the stringent response machinery in living bacterial cells

    PNAS 108:E365–E373.

    https://doi.org/10.1073/pnas.1102255108

    • Google Scholar
26. 1. Ferullo DJ
    2. Lovett ST

    (2008) The stringent response and cell cycle arrest in Escherichia coli

    PLoS Genetics 4:e1000300.

    https://doi.org/10.1371/journal.pgen.1000300

    • Google Scholar
27. 1. Fischer N
    2. Neumann P
    3. Konevega AL
    4. Bock LV
    5. Ficner R
    6. Rodnina MV
    7. Stark H

    (2015) Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

    Nature 520:567–570.

    https://doi.org/10.1038/nature14275

    • Google Scholar
28. Data

    1. Forouhar F
    2. Lew S
    3. Seetharaman J
    4. Sahdev S
    5. Xiao R
    6. Ciccosanti C
    7. Lee D
    8. Everett JK
    9. Nair R
    10. Acton TB
    11. Rost B
    12. Montelione GT
    13. Hunt JF
    14. Tong L
    15. Northeast Structural Genomics Consortium

    (authors) (2009)

    Crystal structure of the TGS domain of the CLOLEP_03100 protein from clostridium leptum, northeast structural genomics consortium target QlR13A

    RCSB Protein Data Bank.
29. 1. Frank J
    2. Agrawal RK

    (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation

    Nature 406:318–322.

    https://doi.org/10.1038/35018597

    • Google Scholar
30. 1. Frank J
    2. Radermacher M
    3. Zhu J
    4. Zhu J
    5. Li Y
    6. Ladjadj M
    7. Leith A

    (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields

    Journal of Structural Biology 116:190–199.

    https://doi.org/10.1006/jsbi.1996.0030

    • Google Scholar
31. 1. Friesen JD
    2. Fiil NP
    3. Parker JM
    4. Haseltine WA

    (1974) A new relaxed mutant of Escherichia coli with an altered 50S ribosomal subunit

    PNAS 71:3465–3469.

    https://doi.org/10.1073/pnas.71.9.3465

    • Google Scholar
32. 1. Gabashvili IS
    2. Agrawal RK
    3. Spahn CM
    4. Grassucci RA
    5. Svergun DI
    6. Frank J
    7. Penczek P

    (2000) Solution structure of the E. coli 70S ribosome at 11.5 Å resolution

    Cell 100:537–549.

    https://doi.org/10.1016/S0092-8674(00)80690-X

    • Google Scholar
33. 1. Gao P
    2. Ascano M
    3. Wu Y
    4. Barchet W
    5. Gaffney BL
    6. Zillinger T
    7. Serganov AA
    8. Liu Y
    9. Jones RA
    10. Hartmann G
    11. Tuschl T
    12. Patel DJ

    (2013) Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase

    Cell 153:1094–1107.

    https://doi.org/10.1016/j.cell.2013.04.046

    • Google Scholar
34. 1. Gao W
    2. Chua K
    3. Davies JK
    4. Newton HJ
    5. Seemann T
    6. Harrison PF
    7. Holmes NE
    8. Rhee HW
    9. Hong JI
    10. Hartland EL
    11. Stinear TP
    12. Howden BP

    (2010) Two novel point mutations in clinical staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection

    PLoS Pathogens 6:e1000944.

    https://doi.org/10.1371/journal.ppat.1000944

    • Google Scholar
35. 1. Gao YG
    2. Selmer M
    3. Dunham CM
    4. Weixlbaumer A
    5. Kelley AC
    6. Ramakrishnan V

    (2009) The structure of the ribosome with elongation factor G trapped in the posttranslocational state

    Science 326:694–699.

    https://doi.org/10.1126/science.1179709

    • Google Scholar
36. 1. Gentry D
    2. Li T
    3. Rosenberg M
    4. McDevitt D

    (2000) The rel gene is essential for in vitro growth of Staphylococcus aureus

    Journal of Bacteriology 182:4995–4997.

    https://doi.org/10.1128/JB.182.17.4995-4997.2000

    • Google Scholar
37. 1. Gonen T
    2. Cheng Y
    3. Sliz P
    4. Hiroaki Y
    5. Fujiyoshi Y
    6. Harrison SC
    7. Walz T

    (2005) Lipid-protein interactions in double-layered two-dimensional AQP0 crystals

    Nature 438:633–638.

    https://doi.org/10.1038/nature04321

    • Google Scholar
38. 1. Gonzalez RL
    2. Chu S
    3. Puglisi JD

    (2007) Thiostrepton inhibition of tRNA delivery to the ribosome

    RNA 13:2091–2097.

    https://doi.org/10.1261/rna.499407

    • Google Scholar
39. 1. Grant GA

    (2006) The ACT domain: a small molecule binding domain and its role as a common regulatory element

    Journal of Biological Chemistry 281:33825–33829.

    https://doi.org/10.1074/jbc.R600024200

    • Google Scholar
40. 1. Grant T
    2. Grigorieff N

    (2015) Automatic estimation and correction of anisotropic magnification distortion in electron microscopes

    Journal of Structural Biology 192:204–208.

    https://doi.org/10.1016/j.jsb.2015.08.006

    • Google Scholar
41. 1. Grant T
    2. Grigorieff N

    (2015b) Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6

    eLife 4:e06980.

    https://doi.org/10.7554/eLife.06980

    • Google Scholar
42. 1. Greber BJ
    2. Boehringer D
    3. Leibundgut M
    4. Bieri P
    5. Leitner A
    6. Schmitz N
    7. Aebersold R
    8. Ban N

    (2014) The complete structure of the large subunit of the mammalian mitochondrial ribosome

    Nature 515:283–286.

    https://doi.org/10.1038/nature13895

    • Google Scholar
43. 1. Grigorieff N

    (2016)

    Methods in Enzymology

     Frealign: An Exploratory Tool for Single-Particle Cryo-EM, Methods in Enzymology, Academic Press.

    • Google Scholar
44. 1. Gropp M
    2. Strausz Y
    3. Gross M
    4. Glaser G

    (2001) Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain

    Journal of Bacteriology 183:570–579.

    https://doi.org/10.1128/JB.183.2.570-579.2001

    • Google Scholar
45. 1. Haseltine WA
    2. Block R
    3. Gilbert W
    4. Weber K

    (1972) MSI and MSII made on ribosome in idling step of protein synthesis

    Nature 238:381–384.

    https://doi.org/10.1038/238381a0

    • Google Scholar
46. 1. Haseltine WA
    2. Block R

    (1973) Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes

    PNAS 70:1564–1568.

    https://doi.org/10.1073/pnas.70.5.1564

    • Google Scholar
47. 1. Hogg T
    2. Mechold U
    3. Malke H
    4. Cashel M
    5. Hilgenfeld R

    (2004) Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]

    Cell 117:57–68.

    https://doi.org/10.1016/S0092-8674(04)00260-0

    • Google Scholar
48. 1. Holm L
    2. Rosenström P

    (2010) Dali server: conservation mapping in 3D

    Nucleic Acids Research 38:W545–W549.

    https://doi.org/10.1093/nar/gkq366

    • Google Scholar
49. 1. Jain V
    2. Kumar M
    3. Chatterji D

    (2006a)

    ppGpp: stringent response and survival

    Journal of Microbiology 44:1–10.

    • Google Scholar
50. 1. Jain V
    2. Saleem-Batcha R
    3. China A
    4. Chatterji D

    (2006b) Molecular dissection of the mycobacterial stringent response protein Rel

    Protein Science 15:1449–1464.

    https://doi.org/10.1110/ps.062117006

    • Google Scholar
51. 1. Jenner L
    2. Demeshkina N
    3. Yusupova G
    4. Yusupov M

    (2010) Structural rearrangements of the ribosome at the tRNA proofreading step

    Nature Structural & Molecular Biology 17:1072–1078.

    https://doi.org/10.1038/nsmb.1880

    • Google Scholar
52. 1. Jenvert RM
    2. Schiavone LH

    (2007) The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

    Journal of Molecular Biology 365:764–772.

    https://doi.org/10.1016/j.jmb.2006.10.065

    • Google Scholar
53. 1. Justesen J
    2. Lund T
    3. Skou Pedersen F
    4. Kjeldgaard NO

    (1986) The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli

    Biochimie 68:715–722.

    https://doi.org/10.1016/S0300-9084(86)80165-1

    • Google Scholar
54. 1. Kim DE
    2. Chivian D
    3. Baker D

    (2004) Protein structure prediction and analysis using the Robetta server

    Nucleic Acids Research 32:W526–W531.

    https://doi.org/10.1093/nar/gkh468

    • Google Scholar
55. 1. Knutsson Jenvert RM
    2. Holmberg Schiavone L

    (2005) Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli

    FEBS Journal 272:685–695.

    https://doi.org/10.1111/j.1742-4658.2004.04502.x

    • Google Scholar
56. 1. Korostelev A
    2. Asahara H
    3. Lancaster L
    4. Laurberg M
    5. Hirschi A
    6. Zhu J
    7. Trakhanov S
    8. Scott WG
    9. Noller HF

    (2008) Crystal structure of a translation termination complex formed with release factor RF2

    PNAS 105:19684–19689.

    https://doi.org/10.1073/pnas.0810953105

    • Google Scholar
57. 1. Korostelev A
    2. Bertram R
    3. Chapman MS

    (2002) Simulated-annealing real-space refinement as a tool in model building

    Acta Crystallographica Section D Biological Crystallography 58:761–767.

    https://doi.org/10.1107/S0907444902003402

    • Google Scholar
58. 1. Kranzusch PJ
    2. Lee AS
    3. Berger JM
    4. Doudna JA
    5. Lee AS-Y
    6. Jennifer A

    (2013) Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity

    Cell Reports 3:1362–1368.

    https://doi.org/10.1016/j.celrep.2013.05.008

    • Google Scholar
59. 1. Kranzusch PJ
    2. Lee AS
    3. Wilson SC
    4. Solovykh MS
    5. Vance RE
    6. Berger JM
    7. Doudna JA
    8. Jennifer A

    (2014) Structure-guided reprogramming of human cGAS dinucleotide linkage specificity

    Cell 158:1011–1021.

    https://doi.org/10.1016/j.cell.2014.07.028

    • Google Scholar
60. 1. Kremer JR
    2. Mastronarde DN
    3. McIntosh JR

    (1996) Computer visualization of three-dimensional image data using IMOD

    Journal of Structural Biology 116:71–76.

    https://doi.org/10.1006/jsbi.1996.0013

    • Google Scholar
61. 1. Kuroda A

    (2006) A polyphosphate-lon protease complex in the adaptation of Escherichia coli to amino acid starvation

    Bioscience, Biotechnology, and Biochemistry 70:325–331.

    https://doi.org/10.1271/bbb.70.325

    • Google Scholar
62. 1. Laurberg M
    2. Asahara H
    3. Korostelev A
    4. Zhu J
    5. Trakhanov S
    6. Noller HF

    (2008) Structural basis for translation termination on the 70S ribosome

    Nature 454:852–857.

    https://doi.org/10.1038/nature07115

    • Google Scholar
63. 1. Lee MS
    2. Gippert GP
    3. Soman KV
    4. Case DA
    5. Wright PE

    (1989) Three-dimensional solution structure of a single zinc finger DNA-binding domain

    Science 245:635–637.

    https://doi.org/10.1126/science.2503871

    • Google Scholar
64. 1. Li W
    2. Bouveret E
    3. Zhang Y
    4. Liu K
    5. Wang JD
    6. Weisshaar JC

    (2016) Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli

    Molecular Microbiology 99:571–585.

    https://doi.org/10.1111/mmi.13252

    • Google Scholar
65. 1. Lohöfener J
    2. Steinke N
    3. Kay-Fedorov P
    4. Baruch P
    5. Nikulin A
    6. Tishchenko S
    7. Manstein DJ
    8. Fedorov R

    (2015) The Activation Mechanism of 2'-5'-Oligoadenylate Synthetase Gives New Insights Into OAS/cGAS Triggers of Innate Immunity

    Structure 23:851–862.

    https://doi.org/10.1016/j.str.2015.03.012

    • Google Scholar
66. 1. Lyumkis D
    2. Brilot AF
    3. Theobald DL
    4. Grigorieff N

    (2013) Likelihood-based classification of cryo-EM images using FREALIGN

    Journal of Structural Biology 183:377–388.

    https://doi.org/10.1016/j.jsb.2013.07.005

    • Google Scholar
67. 1. Magnusson LU
    2. Farewell A
    3. Nyström T

    (2005) ppGpp: a global regulator in Escherichia coli

    Trends in Microbiology 13:236–242.

    https://doi.org/10.1016/j.tim.2005.03.008

    • Google Scholar
68. 1. Mastronarde DN

    (2005) Automated electron microscope tomography using robust prediction of specimen movements

    Journal of Structural Biology 152:36–51.

    https://doi.org/10.1016/j.jsb.2005.07.007

    • Google Scholar
69. 1. Mechold U
    2. Murphy H
    3. Brown L
    4. Cashel M

    (2002) Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis

    Journal of Bacteriology 184:2878–2888.

    https://doi.org/10.1128/JB.184.11.2878-2888.2002

    • Google Scholar
70. 1. Mindell JA
    2. Grigorieff N

    (2003) Accurate determination of local defocus and specimen tilt in electron microscopy

    Journal of Structural Biology 142:334–347.

    https://doi.org/10.1016/S1047-8477(03)00069-8

    • Google Scholar
71. 1. Mittenhuber G

    (2001)

    Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins)

    Journal of Molecular Microbiology and Biotechnology 3:585–600.

    • Google Scholar
72. 1. Moazed D
    2. Noller HF

    (1986) Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes

    Cell 47:985–994.

    https://doi.org/10.1016/0092-8674(86)90813-5

    • Google Scholar
73. 1. Moazed D
    2. Noller HF

    (1989) Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites

    Cell 57:585–597.

    https://doi.org/10.1016/0092-8674(89)90128-1

    • Google Scholar
74. 1. Na HS
    2. Kim HJ
    3. Lee HC
    4. Hong Y
    5. Rhee JH
    6. Choy HE

    (2006) Immune response induced by Salmonella typhimurium defective in ppGpp synthesis

    Vaccine 24:2027–2034.

    https://doi.org/10.1016/j.vaccine.2005.11.031

    • Google Scholar
75. 1. Neidhardt FC

    (1987)

    Escherichia Coli and Salmonella Typhimurium

    Cellular and Molecular Biology, Escherichia Coli and Salmonella Typhimurium, Vol 1-2, American Society for Microbiology.

    • Google Scholar
76. 1. Nguyen D
    2. Joshi-Datar A
    3. Lepine F
    4. Bauerle E
    5. Olakanmi O
    6. Beer K
    7. McKay G
    8. Siehnel R
    9. Schafhauser J
    10. Wang Y
    11. Britigan BE
    12. Singh PK

    (2011) Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria

    Science 334:982–986.

    https://doi.org/10.1126/science.1211037

    • Google Scholar
77. 1. Ogle JM
    2. Brodersen DE
    3. Clemons WM
    4. Tarry MJ
    5. Carter AP
    6. Ramakrishnan V

    (2001) Recognition of cognate transfer RNA by the 30S ribosomal subunit

    Science 292:897–902.

    https://doi.org/10.1126/science.1060612

    • Google Scholar
78. 1. Ogle JM
    2. Carter AP
    3. Ramakrishnan V

    (2003) Insights into the decoding mechanism from recent ribosome structures

    Trends in Biochemical Sciences 28:259–266.

    https://doi.org/10.1016/S0968-0004(03)00066-5

    • Google Scholar
79. 1. Ogle JM
    2. Murphy FV
    3. Tarry MJ
    4. Ramakrishnan V

    (2002) Selection of tRNA by the ribosome requires a transition from an open to a closed form

    Cell 111:721–732.

    https://doi.org/10.1016/S0092-8674(02)01086-3

    • Google Scholar
80. 1. Pape T
    2. Wintermeyer W
    3. Rodnina MV

    (1998) Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome

    The EMBO Journal 17:7490–7497.

    https://doi.org/10.1093/emboj/17.24.7490

    • Google Scholar
81. 1. Parker J
    2. Watson RJ
    3. Friesen JD
    4. Fiil NP

    (1976) A relaxed mutant with an altered ribosomal protein L11

    MGG Molecular & General Genetics 144:111–114.

    https://doi.org/10.1007/BF00277313

    • Google Scholar
82. 1. Pedersen FS
    2. Kjeldgaard NO

    (1977) Analysis of the relA gene product of Escherichia coli

    European Journal of Biochemistry 76:91–97.

    https://doi.org/10.1111/j.1432-1033.1977.tb11573.x

    • Google Scholar
83. 1. Pettersen EF
    2. Goddard TD
    3. Huang CC
    4. Couch GS
    5. Greenblatt DM
    6. Meng EC
    7. Ferrin TE

    (2004) UCSF Chimera--a visualization system for exploratory research and analysis

    Journal of Computational Chemistry 25:1605–1612.

    https://doi.org/10.1002/jcc.20084

    • Google Scholar
84. 1. Pizarro-Cerdá J
    2. Tedin K

    (2004) The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression

    Molecular Microbiology 52:1827–1844.

    https://doi.org/10.1111/j.1365-2958.2004.04122.x

    • Google Scholar
85. 1. Polakis SE
    2. Guchhait RB
    3. Lane MD

    (1973)

    Stringent control of fatty acid synthesis in Escherichia coli. Possible regulation of acetyl coenzyme A carboxylase by ppGpp

     Journal of Biological Chemistry 248:7957–7966.

    • Google Scholar
86. 1. Potrykus K
    2. Cashel M

    (2008) (p)ppGpp: still magical?

    Annual Review of Microbiology 62:35–51.

    https://doi.org/10.1146/annurev.micro.62.081307.162903

    • Google Scholar
87. 1. Ramagopal S
    2. Davis BD

    (1974) Localization of the stringent protein of Escherichia coli on the 50S ribosomal subunit

    PNAS 71:820–824.

    https://doi.org/10.1073/pnas.71.3.820

    • Google Scholar
88. 1. Richter D
    2. Nowak P
    3. Kleinert U

    (1975) Escherichia coli stringent factor binds to ribosomes at a site different from that of elongation factor Tu or G

    Biochemistry 14:4414–4420.

    https://doi.org/10.1021/bi00691a012

    • Google Scholar
89. 1. Richter D

    (1976) Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA

    PNAS 73:707–711.

    https://doi.org/10.1073/pnas.73.3.707

    • Google Scholar
90. 1. Rodnina MV
    2. Fricke R
    3. Kuhn L
    4. Wintermeyer W

    (1995)

    Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome

    The EMBO Journal 14:2613–2619.

    • Google Scholar
91. 1. Rodnina MV
    2. Fricke R
    3. Wintermeyer W

    (1994) Transient conformational states of aminoacyl-tRNA during ribosome binding catalyzed by elongation factor Tu

    Biochemistry 33:12267–12275.

    https://doi.org/10.1021/bi00206a033

    • Google Scholar
92. 1. Rodnina MV
    2. Pape T
    3. Fricke R
    4. Kuhn L
    5. Wintermeyer W

    (1996) Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome

    Journal of Biological Chemistry 271:646–652.

    https://doi.org/10.1074/jbc.271.2.646

    • Google Scholar
93. 1. Sankaranarayanan R
    2. Dock-Bregeon AC
    3. Romby P
    4. Caillet J
    5. Springer M
    6. Rees B
    7. Ehresmann C
    8. Ehresmann B
    9. Moras D

    (1999) The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site

    Cell 97:371–381.

    https://doi.org/10.1016/S0092-8674(00)80746-1

    • Google Scholar
94. 1. Schmeing TM
    2. Voorhees RM
    3. Kelley AC
    4. Gao YG
    5. Murphy FV
    6. Weir JR
    7. Ramakrishnan V

    (2009) The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA

    Science 326:688–694.

    https://doi.org/10.1126/science.1179700

    • Google Scholar
95. 1. Schreiber G
    2. Metzger S
    3. Aizenman E
    4. Roza S
    5. Cashel M
    6. Glaser G

    (1991)

    Overexpression of the relA gene in Escherichia coli

     Journal of Biological Chemistry 266:3760–3767.

    • Google Scholar
96. 1. Selmer M
    2. Dunham CM
    3. Murphy FV
    4. Weixlbaumer A
    5. Petry S
    6. Kelley AC
    7. Weir JR
    8. Ramakrishnan V

    (2006) Structure of the 70S ribosome complexed with mRNA and tRNA

    Science 313:1935–1942.

    https://doi.org/10.1126/science.1131127

    • Google Scholar
97. 1. Shyp V
    2. Tankov S
    3. Ermakov A
    4. Kudrin P
    5. English BP
    6. Ehrenberg M
    7. Tenson T
    8. Elf J
    9. Hauryliuk V

    (2012) Positive allosteric feedback regulation of the stringent response enzyme RelA by its product

    EMBO Reports 13:835–839.

    https://doi.org/10.1038/embor.2012.106

    • Google Scholar
98. 1. Stark H
    2. Dube P
    3. Lührmann R
    4. Kastner B

    (2001) Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle

    Nature 409:539–542.

    https://doi.org/10.1038/35054102

    • Google Scholar
99. 1. Stark H
    2. Rodnina MV
    3. Wieden H-J
    4. Zemlin F
    5. Wintermeyer W
    6. van Heel M

    (2002) Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex

    Nature Structural Biology 9:849–854.

    https://doi.org/10.1038/nsb859

    • Google Scholar
100. 1. Steinchen W
     2. Schuhmacher JS
     3. Altegoer F
     4. Fage CD
     5. Srinivasan V
     6. Linne U
     7. Marahiel MA
     8. Bange G

     (2015) Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

     PNAS 112:13348–13353.

     https://doi.org/10.1073/pnas.1505271112

     • Google Scholar
101. 1. Sun L
     2. Wu J
     3. Du F
     4. Chen X
     5. Chen ZJ

     (2013) Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway

     Science 339:786–791.

     https://doi.org/10.1126/science.1232458

     • Google Scholar
102. 1. Sun W
     2. Roland KL
     3. Branger CG
     4. Kuang X
     5. Curtiss R

     (2009) The role of relA and spoT in Yersinia pestis KIM5 pathogenicity

     PloS One 4:e6720.

     https://doi.org/10.1371/journal.pone.0006720

     • Google Scholar
103. 1. Sy J
     2. Lipmann F

     (1973) Identification of the synthesis of guanosine tetraphosphate (MS I) as insertion of a pyrophosphoryl group into the 3'-position in guanosine 5'-diphosphate

     PNAS 70:306–309.

     https://doi.org/10.1073/pnas.70.2.306

     • Google Scholar
104. 1. Traxler MF
     2. Summers SM
     3. Nguyen HT
     4. Zacharia VM
     5. Hightower GA
     6. Smith JT
     7. Conway T

     (2008) The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli

     Molecular Microbiology 68:1128–1148.

     https://doi.org/10.1111/j.1365-2958.2008.06229.x

     • Google Scholar
105. 1. Valle M
     2. Sengupta J
     3. Swami NK
     4. Grassucci RA
     5. Burkhardt N
     6. Nierhaus KH
     7. Agrawal RK
     8. Frank J

     (2002) Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

     The EMBO Journal 21:3557–3567.

     https://doi.org/10.1093/emboj/cdf326

     • Google Scholar
106. 1. van Heel M
     2. Harauz G
     3. Orlova EV
     4. Schmidt R
     5. Schatz M

     (1996) A new generation of the IMAGIC image processing system

     Journal of Structural Biology 116:17–24.

     https://doi.org/10.1006/jsbi.1996.0004

     • Google Scholar
107. 1. Vogt SL
     2. Green C
     3. Stevens KM
     4. Day B
     5. Erickson DL
     6. Woods DE
     7. Storey DG

     (2011) The stringent response is essential for Pseudomonas aeruginosa virulence in the rat lung agar bead and Drosophila melanogaster feeding models of infection

     Infection and Immunity 79:4094–4104.

     https://doi.org/10.1128/IAI.00193-11

     • Google Scholar
108. 1. Voorhees RM
     2. Schmeing TM
     3. Kelley AC
     4. Ramakrishnan V

     (2010) The mechanism for activation of GTP hydrolysis on the ribosome

     Science 330:835–838.

     https://doi.org/10.1126/science.1194460

     • Google Scholar
109. 1. Voorhees RM
     2. Weixlbaumer A
     3. Loakes D
     4. Kelley AC
     5. Ramakrishnan V

     (2009) Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

     Nature Structural & Molecular Biology 16:528–533.

     https://doi.org/10.1038/nsmb.1577

     • Google Scholar
110. 1. Wagner EG
     2. Kurland CG

     (1980) Escherichia coli elongation factor G blocks stringent factor

     Biochemistry 19:1234–1240.

     https://doi.org/10.1021/bi00547a030

     • Google Scholar
111. 1. Wang JD
     2. Sanders GM
     3. Grossman AD

     (2007) Nutritional control of elongation of DNA replication by (p)ppGpp

     Cell 128:865–875.

     https://doi.org/10.1016/j.cell.2006.12.043

     • Google Scholar
112. 1. Wendrich TM
     2. Blaha G
     3. Wilson DN
     4. Marahiel MA
     5. Nierhaus KH

     (2002) Dissection of the mechanism for the stringent factor RelA

     Molecular Cell 10:779–788.

     https://doi.org/10.1016/S1097-2765(02)00656-1

     • Google Scholar
113. 1. Xu D
     2. Zhang Y

     (2012) Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field

     Proteins 80:1715–1735.

     https://doi.org/10.1002/prot.24065

     • Google Scholar
114. 1. Yang J
     2. Yan R
     3. Roy A
     4. Xu D
     5. Poisson J
     6. Zhang Y

     (2015) The I-TASSER Suite: protein structure and function prediction

     Nature Methods 12:7–8.

     https://doi.org/10.1038/nmeth.3213

     • Google Scholar
115. 1. Yang X
     2. Ishiguro EE

     (2001a) Dimerization of the RelA protein of Escherichia coli

     Biochemistry and Cell Biology 79:729–736.

     https://doi.org/10.1139/o01-144

     • Google Scholar
116. 1. Yang X
     2. Ishiguro EE

     (2001b) Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

     Journal of Bacteriology 183:6532–6537.

     https://doi.org/10.1128/JB.183.22.6532-6537.2001

     • Google Scholar
117. 1. Yusupov MM
     2. Yusupova GZ
     3. Baucom A
     4. Lieberman K
     5. Earnest TN
     6. Cate JH
     7. Noller HF

     (2001) Crystal structure of the ribosome at 5.5 A resolution

     Science 292:883–896.

     https://doi.org/10.1126/science.1060089

     • Google Scholar
118. 1. Zhou G
     2. Wang J
     3. Blanc E
     4. Chapman MS

     (1998) Determination of the relative precision of atoms in a macromolecular structure

     Acta Crystallographica Section D Biological Crystallography 54:391–399.

     https://doi.org/10.1107/S0907444997011530

     • Google Scholar

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

• 5,228

  Page views

• 898

  Downloads

• 110

  Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.