Structural basis of the translational elongation cycle.

The sequential addition of amino acids to a growing polypeptide chain is carried out by the ribosome in a complicated multistep process called the elongation cycle. It involves accurate selection of each aminoacyl tRNA as dictated by the mRNA codon, catalysis of peptide bond formation, and movement of the tRNAs and mRNA through the ribosome. The process requires the GTPase factors elongation factor Tu (EF-Tu) and EF-G. Not surprisingly, large conformational changes in both the ribosome and its tRNA substrates occur throughout protein elongation. Major advances in our understanding of the elongation cycle have been made in the past few years as a result of high-resolution crystal structures that capture various states of the process, as well as biochemical and computational studies.

Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail.

Common chaperone activity in the G-domain of trGTPase protects L11-L12 interaction on the ribosome.

Translational GTPases (trGTPases) regulate all phases of protein synthesis. An early event in the interaction of a trGTPase with the ribosome is the contact of the G-domain with the C-terminal domain (CTD) of ribosomal protein L12 (L12-CTD) and subsequently interacts with the N-terminal domain of L11 (L11-NTD). However, the structural and functional relationships between L12-CTD and L11-NTD remain unclear. Here, we performed mutagenesis, biochemical and structural studies to identify the interactions between L11-NTD and L12-CTD. Mutagenesis of conserved residues in the interaction site revealed their role in the docking of trGTPases. During docking, loop62 of L11-NTD protrudes into a cleft in L12-CTD, leading to an open conformation of this domain and exposure of hydrophobic core. This unfavorable situation for L12-CTD stability is resolved by a chaperone-like activity of the contacting G-domain. Our results suggest that all trGTPases-regardless of their different specific functions-use a common mechanism for stabilizing the L11-NTD•L12-CTD interactions.

Consensus among flexible fitting approaches improves the interpretation of cryo-EM data.

Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data.

Elongation factor Tu: a regulatory GTPase with an integrated effector.

Several elongation factors involved in protein synthesis are GTPases that share structural and mechanistic homology with the large family of proteins including Ras and heterotrimeric receptor-coupled G proteins. The structure of elongation factor Tu (EF-Tu) from thermophilic bacteria, in its 'active' GTP-bound form, has recently been solved by X-ray crystallography. Comparison of this structure with the structure of Escherichia coli EF-Tu bound to GDP reveals a dramatic conformational change that is dependent on GTPase activity. The mechanism of this conformational change and of GTPase activation are discussed, and a model for the EF-Tu-GTP complex with aminoacyl-tRNA is presented.

Initial Cluster Analysis.

We study a simple abstract problem motivated by a variety of applications in protein sequence analysis. Consider a string of 0s and 1s of length L, and containing D 1s. If we believe that some or all of the 1s may be clustered near the start of the sequence, which subset is the most significantly so clustered, and how significant is this clustering? We approach this question using the minimum description length principle and illustrate its application by analyzing residues that distinguish translational initiation and elongation factor guanosine triphosphatases (GTPases) from other P-loop GTPases. Within a structure of yeast elongation factor 1[Formula: see text], these residues form a significant cluster centered on a region implicated in guanine nucleotide exchange. Various biomedical questions may be cast as the abstract problem considered here.

Imprinting through molecular mimicry. Protein synthesis.

Part of the structure of translational elongation factor G, in a complex with GDP, resembles the tRNA bound in a ternary complex with elongation factor Tu and GTP; this 'molecular mimicry' extends to charge distribution as well as shape.

Regulatory GTPases.

The past year has witnessed a tremendous increase in our understanding of the structures and interactions of the GTPases. The highlights include crystal structures of G alpha subunits, as well as the first complex between a GTPase (Rap1A) and an effector molecule (c-Raf1 Ras-binding domain). In the field of elongation factors (EFs), three very important structures have been determined: EF-G, the ternary complex of EF-Tu.GTP with aminoacyl-tRNA, and the EF-Tu.EF-Ts complex.

The ABC of EF-G.

The recently solved crystal structures of Thermus thermophilus elongation factor G, with and without GDP, reveal a protein of five domains with surprising features which can be correlated with biochemical data to suggest probable functional roles.

The structure of elongation factor G in complex with GDP: conformational flexibility and nucleotide exchange.

BACKGROUND: Elongation factor G (EF-G) catalyzes the translocation step of translation. During translocation EF-G passes through four main conformational states: the GDP complex, the nucleotide-free state, the GTP complex, and the GTPase conformation. The first two of these conformations have been previously investigated by crystallographic methods. RESULTS: The structure of EF-G-GDP has been refined at 2.4 A resolution. Comparison with the nucleotide-free structure reveals that, upon GDP release, the phosphate-binding loop (P-loop) adopts a closed conformation. This affects the position of helix CG, the switch II loop and domains II, IV and V. Asp83 has a conformation similar to the conformation of the corresponding residue in the EF-Tu/EF-Ts complex. The magnesium ion is absent in EF-G-GDP. CONCLUSIONS: The results illustrate that conformational changes in the P-loop can be transmitted to other parts of the structure. A comparison of the structures of EF-G and EF-Tu suggests that EF-G, like EF-Tu, undergoes a transition with domain rearrangements. The conformation of EF-G-GDP around the nucleotide-binding site may be related to the mechanism of nucleotide exchange.

An alpha to beta conformational switch in EF-Tu.

BACKGROUND: The bacterial elongation factor EF-Tu recognizes and transports aminoacyl-tRNAs to mRNA-programmed ribosomes. EF-Tu shares many structural and functional properties with other GTPases whose conformations are regulated by guanine nucleotides. RESULTS: An intact form of Escherichia coli EF-Tu complexed with GDP has been crystallized in the presence of the EF-Tu-specific antibiotic GE2270 A. The three-dimensional structure has been solved by X-ray diffraction analysis and refined to a final crystallographic R factor of 17.2% at a resolution of 2.5 A. The location of the GE2270 A antibiotic-binding site could not be identified. CONCLUSIONS: The structure of EF-Tu-GDP is nearly identical to that of a trypsin-modified form of EF-Tu-GDP, demonstrating conclusively that the protease treatment had not altered any essential structural features. The present structure represents the first view of an ordered Switch I region in EF-Tu-GDP and reveals similarities with two other GTPases complexed with GDP: Ran and ADP-ribosylation factor-1. A comparison of the Switch I regions of the GTP and GDP forms of EF-Tu also reveals that a segment, six amino acids in length, completely converts from an alpha helix in the GTP complex to beta secondary structure in the GDP form. The alpha to beta switch in EF-Tu may represent a prototypical activation mechanism for other protein families.

The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA.

BACKGROUND: . The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. RESULTS: The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. CONCLUSIONS: The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.

Classification and evolution of P-loop GTPases and related ATPases.

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.

[GTPases of translational apparatus].

Protein biosynthesis is a complex biochemical process. It integrates multiple steps where different translation factors specifically interact with the ribosome in a precisely defined order. Among the translation factors one can find multiple GTP-binding or G-proteins. Their functioning is accompanied by GTP hydrolysis to the GDP and inorganic phosphate ion Pi. Ribosome stimulates the GTPase activity of the translation factors, thus playing a role analogues to GTPase-activating proteins (GAP). Translation factors--GTPases interact with the ribosome at all stages of protein biosynthesis. Initiation factor 2 (IF2) catalyse initiator tRNA binding to the ribosomal P-site and subsequent subunit joining. Elongation factor Tu (EF-Tu) is responsible for the aminoacyl-tRNA binding to the ribosomal A-site, while elongation factor G (EF-G) catalyses translocation of mRNA in the ribosome by one codon, accompanied by tRNA movement between the binding sites. In its turn, release factor 3 (RF3) catalyse dissociation of the ribosomal complex with release factors 1 or 2 (RF1 or RF2) following the peptide release. This review is devoted to the functional peculiarities of translational GTPases as related to other G-proteins. Particularly, to the putative GTPase activation mechanism, structure and functional cycles.

Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation.

Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.

Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine.

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

[Characteristics of N-terminal 60-kDa fragment of elongation factor 2]. / Svoistva N-kontsevogo 60-kDa fragmenta faktora élongatsii 2.

The N-terminal 60-kDa-fragment of elongation factor 2 from rat liver (EF-2) was obtained by the limited proteolysis of native EF-2 with elastase. This fragment consists of 506 N-terminal amino acid residues of EF-2. The conformational properties of both this fragment and EF-2 in solution were studied by circular dichroism and fluorescent spectroscopy. The contents of secondary structure components in the fragment and in the factor that were deduced from CD measurements agreed well with values predicted from their primary structures. Both proteins were resistant to denaturation with < or = 3 M urea and exhibited cooperative denaturation transitions. Temperature melting also proceeded cooperatively for the fragment and EF-2. Structural properties of the N-terminal 60-kDa-fragment are discussed in comparison with the biochemical characteristics and 3D structure of prokaryotic elongation factor EF-G.

Structural studies of elongation and release factors.

The elongation and termination steps of protein synthesis are controlled by elongation and release factors, respectively. Elongation factors deliver the aminoacyl tRNA to the ribosomal A site, ensuring the elongation of the nascent polypeptide chain by one amino acid at a time, while release factors recognize the stop codons and trigger the release of the polypeptide from the ribosome. Recently, high-resolution crystal structures of ribosomes as well as translation factors on and off the ribosome have contributed a great deal to our understanding of the molecular basis of protein synthesis. This review concentrates on recent developments in our understanding of the elongation and termination steps of protein synthesis, particularly the roles of translation factors and their similarities and differences in the eukaryotic cytosol and prokaryotic systems, through a combination of structural and biochemical studies.

Bayesian adaptive alignment and inference.

Sequence alignment without the specification of gap penalties or a scoring matrix is attained by using Bayesian inference and a recursive algorithm. This procedure's recursive algorithm sums over all possible alignments on the forward step to obtain normalizing constants essential to Bayesian inferences, and samples from the exact posterior distribution on the backward step. Since both terminal and intervening unrelated subsequences will often be excluded from an alignment, the resulting alignments may be seen as extensions of local alignments. An alignment's significance is assessed using the Bayesian evidence. A shuffling simulation shows that Bayesian evidence against the null hypothesis tends to be a conservative measure of significance compared to classical p-values. An application to proteins from the GTPase superfamily shows that the posterior distribution of the number of gaps is often flat and that the posterior distribution of the evolutionary distance is often flat and sometimes bimodal. An alignment of 1GIA with 1ETU shows good correspondence with a structural alignment.