Elongation Factor G Research Articles

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-tRNAs to the A-site of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A-site1,2. The translocation reaction is catalyzed by elongation factor G (EF-G) in a GTP-dependent fashion3. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryo-EM analysis to resolve two previously unseen subpopulations within EF-G-ribosome complexes at sub-nanometer resolution, one of them with a partially translocated tRNA. Comparison of these sub-states reveals that translocation of tRNA on the 30S subunit parallels the swiveling of the 30S-head and is coupled to un-ratcheting of the 30S-body. Since the tRNA maintains contact with the P-site on the 30S-head and simultaneously establishes interaction with the E-site on the 30S-platform, a novel intra-subunit pe/E hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swiveled 30S-head conformation. These findings provide direct structural and mechanistic insight into the “missing link” in terms of tRNA intermediates involved in the universally conserved translocation process.

Evolution of Elongation Factor G and the Origins of Mitochondrial and Chloroplast Forms

Protein synthesis elongation factor G (EF-G) is an essential protein with central roles in both the elongation and ribosome recycling phases of protein synthesis. Although EF-G evolution is predicted to be conservative, recent reports suggest otherwise. We have characterized EF-G in terms of its molecular phylogeny, genomic context, and patterns of amino acid substitution. We find that most bacteria carry a single "canonical" EF-G, which is phylogenetically conservative and encoded in an str operon. However, we also find a number of EF-G paralogs. These include a pair of EF-Gs that are mostly found together and in an eclectic subset of bacteria, specifically δ-proteobacteria, spirochaetes, and planctomycetes (the "spd" bacteria). These spdEFGs have also given rise to the mitochondrial factors mtEFG1 and mtEFG2, which probably arrived in eukaryotes before the eukaryotic last common ancestor. Meanwhile, chloroplasts apparently use an α-proteobacterial-derived EF-G rather than the expected cyanobacterial form. The long-term comaintenance of the spd/mtEFGs may be related to their subfunctionalization for translocation and ribosome recycling. Consistent with this, patterns of sequence conservation and site-specific evolutionary rate shifts suggest that the faster evolving spd/mtEFG2 has lost translocation function, but surprisingly, the protein also shows little conservation of sites related to recycling activity. On the other hand, spd/mtEFG1, although more slowly evolving, shows signs of substantial remodeling. This is particularly extensive in the GTPase domain, including a highly conserved three amino acid insertion in switch I. We suggest that subfunctionalization of the spd/mtEFGs is not a simple case of specialization for subsets of original activities. Rather, the duplication allows the release of one paralog from the selective constraints imposed by dual functionality, thus allowing it to become more highly specialized. Thus, the potential for fine tuning afforded by subfunctionalization may explain the maintenance of EF-G paralogs.

Correlated conformational events in EF-G and the ribosome regulate translocation

In bacteria, the translocation of tRNA and mRNA with respect to the ribosome is catalyzed by the conserved GTPase, elongation factor-G (EF-G). In order to probe the rate determining features in this process, EF-G-catalyzed translocation was imaged from two unique structural perspectives using single-molecule fluorescence resonance energy transfer. The data reveal that the rate at which the ribosome spontaneously achieves a transient, “unlocked” state is closely correlated with the rate at which the tRNA-like, domain IV/V element of EF-G engages the A site. Following these structural transitions, translocation occurs comparatively fast, suggesting that conformational processes intrinsic to the ribosome determine the rate of translocation. Experiments performed in the presence of non-hydrolyzable GTP analogues and specific antibiotics further reveal that allosterically linked conformational events in EF-G and the ribosome mediate rapid, directional substrate movement and EF-G release.

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5–25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7–2.8 μM Kdversus 9.1–13.9 μM Kd at 10–25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNAfMet demonstrating that IF2 binds to initiator tRNA with submicromolar Kd and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNAfMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNAfMet binding and GTP was suggested to strongly promote fMet-tRNAfMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNAfMet selection.

Staphylococcus aureus elongation factor G – structure and analysis of a target for fusidic acid

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 Å resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 Å relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.

Origin of the Y genome in Elymus and its relationship to other genomes in Triticeae based on evidence from elongation factor G ( EF-G) gene sequences

It is well known that Elymus arose through hybridization between representatives of different genera. Cytogenetic analyses show that all its members include the St genome in combination with one or more of four other genomes, the H, Y, P, and W genomes. The origins of the H, P, and W genomes are known, but not for the Y genome. We analyzed the single copy nuclear gene coding for elongation factor G ( EF-G) from 28 accessions of polyploid Elymus species and 45 accessions of diploid Triticeae species in order to investigate origin of the Y genome and its relationship to other genomes in the tribe Triticeae. Sequence comparisons among the St, H, Y, P, W, and E genomes detected genome-specific polymorphisms at 66 nucleotide positions. The St and Y genomes are relatively dissimilar. The phylogeny of the Y genome sequences was investigated for the first time. They were most similar to the W genome sequences. The Y genome sequences were placed in two different groups. These two groups were included in an unresolved clade that included the W and E sequences as well as sequences from many annual species. The H genomes sequences were in a clade with the F, P, and Ns genome sequences as sister groups. These two clades were more closely related to each other and to the L and Xp genomes than they were to the St genome sequences. These data support the hypothesis that the Y genome evolved in a diploid species and has a different origin from the St genome.

The dynamic view of the ribosome in translocation

During the translocation step of protein synthesis, two transfer RNAs (tRNAs) move by distances of 20 Å or more, to their adjacent sites in the ribosome. Elongation factor G (EF‐G) catalyzes translocation, hydrolyzing GTP in the process. Translocation can proceed spontaneously in both forward and backward directions implying that it is a function inherent to the ribosome itself. The movement of the tRNAs is coupled to the conformational changes of the ribosome, which include the ratchet‐like movement of the 30S subunit relative to the 50S subunit, swivel‐like movement of the 30S subunit head, as well as opening of the tRNA binding cleft. The role of EF‐G is to accelerate formation of the “unlocked” state of the ribosome allowing for rapid movement of tRNAs on the 30S subunit, and to prevent the backward movement of the tRNAs after translocation by coupling the energy of GTP hydrolysis to forward translocation. Thus, EF‐G acts as a Brownian ratchet which rectifies the spontaneous thermal motions of the tRNAs within the ribosome into forward translocation.

Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation

Following ribosomal peptide bond formation, the reaction products, peptidyl-tRNA and deacylated tRNA, need to be translocated from the A- and P-sites to the P- and E-sites, respectively. This process is facilitated by the GTPase elongation factor G (EF-G). The mechanism describing how the ribosome activates GTP hydrolysis is poorly understood in molecular terms. By using an 'atomic mutagenesis' approach, which allows the manipulation of specific functional groups on 23S rRNA nucleotides in the context of the entire ribosome, we disclose the adenine exocyclic N6 amino group at A2660 of the sarcin-ricin loop as a key determinant for triggering GTP hydrolysis on EF-G. We show that the purine pi system-expanding characteristics of the exocyclic functional group at the C6 position of A2660 are essential. We propose that stacking interactions of A2660 with EF-G may act as a molecular trigger to induce repositioning of suspected functional amino acids in EF-G that in turn promote GTP hydrolysis.

Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation.

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF-G) in bacteria plays two distinct roles in different phases of the translation system. EF-G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post-termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF-G are distributed over two different EF-G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF-G is also found in bacteria. Two EF-G paralogues are found in the spirochaete Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-G1 is a translocase, while EF-G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF-G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF-3) is present in the reaction. These results indicate that two B. burgdorferi EF-G paralogues are close relatives to mitochondrial EF-G paralogues rather than the conventional bacterial EF-G, in both their phylogenetic and biochemical features.

Intramolecular Movements in EF-G, Trapped at Different Stages in Its GTP Hydrolytic Cycle, Probed by FRET

Elongation factor G (EF-G) is one of several GTP hydrolytic proteins (GTPases) that cycles repeatedly on and off the ribosome during protein synthesis in bacterial cells. In the functional cycle of EF-G, hydrolysis of guanosine 5′-triphosphate (GTP) is coupled to tRNA–mRNA translocation in ribosomes. GTP hydrolysis induces conformational rearrangements in two switch elements in the G domain of EF-G and other GTPases. These switch elements are thought to initiate the cascade of events that lead to translocation and EF-G cycling between ribosomes. To further define the coupling mechanism, we developed a new fluorescent approach that can detect intramolecular movements in EF-G. We attached a fluorescent probe to the switch I element (sw1) of Escherichia coli EF-G. We monitored the position of the sw1 probe, relative to another fluorescent probe anchored to the GTP substrate or product, by measuring the distance-dependent, Förster resonance energy transfer between the two probes. By analyzing EF-G trapped at five different functional states in its cycle, we could infer the cyclical movements of sw1 within EF-G. Our results provide evidence for conformational changes in sw1, which help to drive the unidirectional EF-G cycle during protein synthesis. More generally, our approach might also serve to define the conformational dynamics of other GTPases with their cellular receptors.

PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

The structure of the ribosome with elongation factor G trapped in the posttranslocational state.

Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu.

Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.

EF-G2mt Is an Exclusive Recycling Factor in Mammalian Mitochondrial Protein Synthesis

Bacterial translation elongation factor G (EF-G) catalyzes translocation during peptide elongation and mediates ribosomal disassembly during ribosome recycling in concert with the ribosomal recycling factor (RRF). Two homologs of EF-G have been identified in mitochondria from yeast to man, EF-G1mt and EF-G2mt. Here, we demonstrate that the dual function of bacterial EF-G is divided between EF-G1mt and EF-G2mt in human mitochondria (RRFmt). EF-G1mt specifically catalyzes translocation, whereas EF-G2mt mediates ribosome recycling with human mitochondrial RRF but lacks translocation activity. Domain swapping experiments suggest that the functional specificity for EF-G2mt resides in domains III and IV. Furthermore, GTP hydrolysis by EF-G2mt is not necessary for ribosomal splitting, in contrast to the bacterial-recycling mode. Because EF-G2mt represents a class of translational GTPase that is involved in ribosome recycling, we propose to rename this factor mitochondrial ribosome recycling factor 2 (RRF2mt).

Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis

The translation elongation factor G (EFG) is encoded by the fusA gene. Several bacteria possess a second fusA-like locus, fusA2 which encodes EFG2. A comparison of EFG and EFG2 from various bacteria reveals that EFG2 preserves domain organization and maintains significant sequence homology with EFG, suggesting that EFG2 may function as an elongation factor. However, with the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like factors has not been investigated. Here, we have characterized EFG2 (MSMEG_6535) from Mycobacterium smegmatis. Expression of EFG2 was detected in stationary phase cultures of M. smegmatis (Msm). Our in vitro studies show that while MsmEFG2 binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of EFGs. Furthermore, unlike MsmEFG (MSMEG_1400), MsmEFG2 failed to rescue an E. coli strain harboring a temperature-sensitive allele of EFG, for its growth at the non-permissive temperature. Subsequent experiments showed that the fusA2 gene could be disrupted in M. smegmatis mc(2)155 with Kan(R) marker. The M. smegmatis fusA2::kan strain was viable and showed growth kinetics similar to that of the parent strain (wild-type for fusA2). However, in the growth competition assays, the disruption of fusA2 was found to confer a fitness disadvantage to M. smegmatis, raising the possibility that EFG2 is of some physiological relevance to mycobacteria.

Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936-947). In the present study, we found that inactivation of EF-G (Slr1463) by H(2)O(2) was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H(2)O(2) and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.

Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA-mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by approximately 20 A), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G*GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.