GTPase activation of elongation factor EF‐Tu by the ribosome during decoding

We have used single‐particle reconstruction in cryo‐electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF‐Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF‐Tu, but before the release of EF‐Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å. Secondary structure elements in tRNA, EF‐Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF‐Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF‐Tu.     

EF—Tumt和EF—Tsmt在不同发育阶段小鼠各组织中的表达分析

线粒体蛋白质翻译延长因子Ｔｕ和Ｔｓ（ｍｉｔｏｃｈｏｎｄｒｉａｌｅｌｏｎｇａｔｉｏｎｆａｃｔｏｒＴｕａｎｄＴｓ,ＥＦＴｕｍｔａｎｄＥＦＴｓｍｔ）是由核基因编码的两个蛋白质,它们的功能和调控对细胞的生长发育有重要意义。采用ＥＦＴｕｍｔ和ＥＦＴｓｍｔ重组蛋白分别制备了抗ＥＦＴｕｍｔ和抗ＥＦＴｓｍｔ特异抗体并以此检测了它们在小鼠不同发育时期心肌、骨骼肌、肝、脑、脾等组织中的表达。蛋白质印迹结果表明ＥＦＴｕｍｔ和ＥＦＴｓｍｔ在各组织中的表达水平不同、有明显的组织差异性,并都受发育的调节。ＥＦＴｕｍｔ在同一发育时期各组织中的表达及随发育的变化趋势与ＥＦＴｓｍｔ基本一致。结果提示ＥＦＴｕｍｔ和ＥＦＴｓｍｔ的表达水平与组织细胞能量代谢水平密切相关,它们不仅在体内以复合体形式发挥作用,其基因表达可能受同一机制的调控。     

Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae

The redox‐regulated chaperone Hsp33 protects bacteria specifically against stress conditions that cause oxidative protein unfolding, such as treatment with bleach or exposure to peroxide at elevated temperatures. To gain insight into the mechanism by which expression of Hsp33 confers resistance to oxidative protein unfolding conditions, we made use of Vibrio cholerae strain O395 lacking the Hsp33 gene hslO. We found that this strain, which is exquisitely bleach‐sensitive, displays a temperature‐sensitive (ts) phenotype during aerobic growth, implying that V. cholerae suffers from oxidative heat stress when cultivated at 43°C. We utilized this phenotype to select for Escherichia coli genes that rescue the ts phenotype of V. cholerae ΔhslO when overexpressed. We discovered that expression of a single protein, the elongation factor EF‐Tu, was sufficient to rescue both the ts and bleach‐sensitive phenotypes of V. cholerae ΔhslO. In vivo studies revealed that V. cholerae EF‐Tu is highly sensitive to oxidative protein degradation in the absence of Hsp33, indicating that EF‐Tu is a vital chaperone substrate of Hsp33 in V. cholerae. These results suggest an ‘essential client protein’ model for Hsp33's chaperone action in Vibrio in which stabilization of a single oxidative stress‐sensitive protein is sufficient to enhance the oxidative stress resistance of the whole organism.     

Interaction of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with elongation factors from

The interactions of guanosine 5′-diphosphate, 2′-(or 3′-) diphosphate(ppGpp) with the polypeptide elongation factors Tu(EF Tu) and G(EF G) have been studied. The data indicate that ppGpp binds with EF Tu to form an EF Tu-ppGpp complex, and inhibits, in a competitive manner, the exchange reaction of Tu-GDP and ³H-GDP. The ribosome-dependent GTPase reaction catalyzed by EF G is also depressed by ppGpp.     

Discrimination between aminoacyl groups on su+ 7 tRNA by elongation factor Tu

The su⁺7 nonsense suppressor of Escherichia coli is a mutant tRNA^Trp that can be aminoacylated with either tryptophan or glutamine. We have compared the ternary complexes of glutaminyl and tryptophanyl-su⁺7 tRNA with elongation factor Tu and GTP. Glutaminyl-su⁺7 tRNA binds more strongly than tryptophanyl-su⁺7 tRNA to EF Tu · GTP. The greatest distinction between the two species of the tRNA is seen in their dissociation rates from the complex, which differ by as much as fivefold. The distinction is affected by pH values around neutrality. These results show that EF Tu can distinguish between two aminoacyl-tRNAs which differ only in the aminoacyl group. The implications for the unusual amino acid specificity of su⁺7 tRNA are discussed.     

Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors.

The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-G] for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycin and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EF1)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.     

A model for the interaction of the G3‐subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet‐tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2‐30S interaction, is positioned between the GTP‐binding G2 and the fMet‐tRNA binding C‐terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β‐sheets with each four anti‐parallel strands, followed by a C‐terminal α‐helix. In line with its role as linker between G3 and subdomain C1, this helix has no well‐defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB‐fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA‐binding modules such as IF2‐C2, IF1 and subdomains II of elongation factors EF‐Tu and EF‐G. Structural comparisons have resulted in a model that describes the interaction between IF2‐G3 and the 30S ribosomal subunit.     

The population genetic test Tajima's D identifies genes encoding pathogen‐associated molecular patterns and other virulence‐related genes in Ralstonia solanacearum

The detection of pathogen‐associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor‐Tu (EF‐Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum–plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non‐neutrally evolving genes, including not only EF‐Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence‐related genes in plant pathogen genomes.     

Roles of elusive translational GTPases come to light and inform on the process of ribosome biogenesis in bacteria

Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF‐Tu, EF‐G and RF3, play well‐known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), conversely, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit respectively. These findings have important implications for ribosome biogenesis in bacteria. Because the GTPase activity of each of these proteins depends on interactions with both ribosomal subunits, some portion of 30S and 50S assembly must occur in the context of the 70S ribosome. In this review, we introduce the trGTPases of bacteria, describe the new functional data on LepA and BipA, and discuss the how these findings shape our current view of ribosome biogenesis in bacteria.     

GTP hydrolysis by EF‐G synchronizes tRNA movement on small and large ribosomal subunits

Elongation factor G (EF‐G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF‐G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF‐G mutants and translocation‐specific antibiotics to investigate timing and energetics of translocation. We show that EF‐G–GTP facilitates synchronous movements of peptidyl‐tRNA on the two subunits into an early post‐translocation state, which resembles a chimeric state identified by structural studies. EF‐G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF‐G. Our results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.     

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori

Background. Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. Method. The total complement of protein from seven strains of H. pylori was resolved by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori‐infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme‐antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide‐mass fingerprinting using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results. Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease β‐subunit UreB; elongation factor EF‐Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. Conclusion. These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF‐Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

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.     

Control of the pattern‐recognition receptor EFR by an ER protein complex in plant immunity

In plant innate immunity, the surface‐exposed leucine‐rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen‐associated molecular patterns EF‐Tu and flagellin, respectively. We identified the Arabidopsis stromal‐derived factor‐2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER‐quality control (ER‐QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER‐QC components by EFR and FLS2 could be linked to N‐glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co‐translational N‐glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2–ERdj3B–BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER‐QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER‐QC and N‐glycosylation components by two closely related receptors.     

Structural and functional diversity of EF‐hand proteins: Evolutionary perspectives

We have classified 865 sequences of EF‐hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF‐lobe (pair of adjacent EF‐hands). They have two or more EF‐lobes; the relative positions of their EF‐lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF‐lobes of these groups indicate that the mutual positions of EF‐lobes were established soon after divergence of an EF‐lobe for each group and before the duplication and fusion of EF‐lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF‐lobe precursor, even if their conformations are similar to the canonical EF‐hand. This is an example of convergent evolution.     

Elongation factor Tu-targeted antibiotics: four different structures, two mechanisms of action

Elongation factor Tu (EF-Tu), the carrier of aa-tRNA to the mRNA-programmed ribosome, is the target of four families of antibiotics of unrelated structure, of which the action is supported by two basic mechanisms. Kirromycin and enacyloxin block EF-Tu.GDP on the ribosome; pulvomycin and GE2270 A inhibit the interaction of EF-Tu.GTP with aa-tRNA. The crystallographic analysis has unveiled the structural background of their actions, explaining how antibiotics of unrelated structures and binding modes and sites can employ similar mechanism of action. The selective similarities and differences of their binding sites and the induced EF-Tu conformations make understand how nature can affect the activities of a complex regulatory enzyme by means of low-molecular compounds, and have proposed a suitable approach for drug design.     

Structural resemblance between the families of bacterial signal-transduction proteins and of G proteins revealed by graph theoretical techniques

The first application of a novel technique for the identification of common folding motifs in proteins is presented. Using techniques derived from graph theory, developed in order to compare secondary structure motifs in proteins, we have established that there is a striking resemblance in the tertiary fold of the Salmonella typhimurium Che Y chemotaxis protein and that of the GDP-binding domain of Escherichia coli elongation factor Tu (EF Tu). These two protein structures are representatives of two major macromolecular classes: CheY is a signal-transduction protein with sequence homologies to a wide range of bacterial proteins involved in regulation of chemotaxis, membrane synthesis and sporulation; whilst EF Tu is one of a family of guanosine-nucleotide-binding proteins which include the ras oncogene proteins and signal-transducing G proteins. The similarity we have found extends far beyond the previously recognized resemblances of each protein's fold to that of a generic nucleotide-binding domain. The lack of significant sequence homology between the two classes of proteins may mean that the common fold of the two proteins constitutes a particularly stable folding motif. However, an alternative possibility is that the strong three-dimensional structural resemblance may be indicative of a remote shared common ancestry between the bacterial signal-transduction proteins and the GDP-binding proteins.     

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium,but not nitrogen‐fixing rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR‐Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.     

Expression pattern of acetylated α‐tubulin in porcine spermatogonia

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated α‐tubulin (Ac‐α‐Tu) is a component of long‐lived, stable microtubules and deacetylation of α‐tubulin enhances cell motility, we hypothesized that acetylation of α‐tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac‐α‐Tu at different stages of testis development was characterized by immunohistochemistry for Ac‐α‐Tu and spermatogonia‐specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac‐α‐Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac‐α‐Tu appeared polarized toward the basement membrane. In adult pig testes, Ac‐α‐Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL?), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac‐α‐Tu. Comparison with the expression pattern of β‐tubulin and tyrosinated α‐tubulin confirmed that only Ac‐α‐Tu is specific to germ cells. The unique pattern of Ac‐α‐Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348–352, 2010. © 2009 Wiley‐Liss, Inc.