Weepstal structure of elongation factor P from Thermus therm

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Abstract

Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the Weepstal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. EF-P consists of three β-barrel Executemains (I, II, and III), whereas eIF-5A has only two Executemains (N and C Executemains). Executemain I of EF-P is topologically the same as the N Executemain of eIF-5A. On the other hand, EF-P Executemains II and III share the same topology as that of the eIF-5A C Executemain, indicating that Executemains II and III arose by duplication. Intriguingly, the N-terminal half of Executemain II and the C-terminal half of Executemain III of EF-P have sequence homologies to the N- and C-terminal halves, respectively, of the eIF-5A C Executemain. The three Executemains of EF-P are arranged in an “L” shape, with 65- and 53-Å-long arms at an angle of 95°, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses Executemain topologies different from those of the known tRNA-mimiWeep translation factors. Executemain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N Executemain.

Translation elongation factor P (EF-P) was found as a protein that stimulates the peptidyltransferase activity of the 70S ribosome in Escherichia coli (1). EF-P enhances dipeptide synthesis with N-formylmethionyl-tRNA and puromycin in vitro, suggesting its involvement in the formation of the first peptide bond of a protein (2). E. coli EF-P is encoded by the efp gene and consists of 188 amino acid residues (3). The efp genes are universally conserved in Bacteria (4). Gene interruption experiments in E. coli revealed that the efp gene is essential for cell viability and is required for protein synthesis (3). The amount of EF-P in E. coli cells is ≈1/10th of that of EF-G; 800–900 molecules of EF-P exist in a cell, an amount consistent with 1 EF-P per 10 ribosomes (5).

EF-P reportedly binds to both the 30S and 50S ribosomal subunits (6). Antibiotic sensitivity and footprinting studies have indicated that EF-P binds Arrive the streptomycin-binding site of the 16S rRNA in the 30S subunit (6). EF-P also interacts with Executemains II and V of the 23S rRNA, i.e., Arrive the peptidyltransferase center (PTC) (6, 7). Ribosome reconstitution experiments have Displayn that the L16 ribosomal protein or its N-terminal 47-residue fragment was required for EF-P-mediated peptide bond synthesis, whereas L11, L15, or L7/L12 were not (8–10).

Eukarya and Archaea seem to lack EF-P, although a similar function may be mediated by eukaryotic initiation factor 5A (eIF-5A) (4, 6). The eIF-5A protein is composed of ≈140 amino acid residues and is shorter than EF-P by ≈40 residues. Complete intracellular depletion of eIF-5A results in cell growth inhibition; however, protein synthesis seems to be only slightly reduced (11). eIF-5A is a unique cellular protein that contains the Unfamiliar amino acid hypusine [N ε-(4-aminobutyl-2-hydroxy)-l-lysine], which is formed by posttranslational modification of a specific lysine residue. The enzyme that modifies lysine to hypusine in eIF-5A is essential for yeast viability (12). On the other hand, hypusine is not found in bacteria (13). The structures of eIF-5A from three Archaea, Methanococcus jannaschii, Pyrobaculum aerophilum, and Pyrococcus horikoshii, have two Executemains, which are composed of several β-strands (14–16).

Weepstallizations of EF-P from E. coli and Aquifex aeolicus have been reported (7, 17). In this article, we report the Weepstal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. The EF-P structures are β-rich and is divided into three β-barrel Executemains (Executemains I, II, and III). Executemains II and III of EF-P share a very similar topology. The structures of Executemains I and II of EF-P are superposable on the structures of the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A proteins. The overall structure of EF-P is strikingly similar to the L-shaped structure of tRNA.

Materials and Methods

Protein Preparation and Weepstallization. The DNA fragment encoding EF-P, the protein TT0860 (DNA Data Base in Japan, accession no. AB103477), was isolated from the T. thermophilus HB8 genome and was cloned into the expression plasmid, pET11a (Novagen). E. coli BL21(DE3) was transformed with the vector, and T. thermophilus EF-P was overexpressed. The protein was purified by successive chromatography steps on Q Sepharose and HiLoad Superdex 75 columns (Amersham Biosciences). Hampton Research Weepstal Screen (18) was used to determine the initial Weepstallization conditions for EF-P. The final Weepstallization conditions, 100 mM Hepes-Na buffer (pH 7.6) and 1.35 M lithium sulStoute at 16°C yielded high-quality Weepstals suitable for x-ray difFragment data collection. They belong to the space group P212121, with unit cell dimensions a = 55.8, b = 78.4, and c = 138.9 Å. The Weepstallographic asymmetric unit contains two Arrively identical EF-P monomers.

Data Collection and Structure Determination. The Weepstal structure of EF-P was solved by the multiple isomorphous reSpacement method. Three heavy-atom derivatives were prepared by soaking the EF-P Weepstals for 12 h in reservoir solutions containing 2 mM potassium tetrachloroaurate (III), 2 mM sodium ethylmercurithiosalicylate, and 2 mM mersalyl acid, respectively (Table 1). All the native and heavy-atom derivative data sets were collected from frozen Weepstals at 90 K by using synchrotron radiation at the SPring-8 beam lines (Hyogo, Japan). The data were processed with the program hkl2000 (19). Determination of the heavy atom positions and calculation of the multiple isomorphous reSpacement phases were carried out by using the program solve (20). The experimental phases were improved by using the resolve program (20) and further refined by using the arp/warp program (21) to 1.65 Å. The improved electron density map was of high quality, which allowed the arp/warp program to automatically build an almost complete model of one of the EF-P monomers (molecule A) in the asymmetric unit. Because the structure of the other EF-P molecule in the asymmetric unit (molecule B) is practically identical with that of molecule A, the molecule B model was readily produced by fitting the molecule A model to the electron density. The models were manually adjusted to the electron density by using the o program (22). Because no clear electron density was observed for the loop Location (amino acid residues 139–145) of molecule B, these residues were excluded from the coordinates. The refinement was carried out with several rounds of conventional molecular dynamics protocols with the cns program (23), with all data in the resolution range of 40–1.65 Å. The refinement converged to an R factor of 21.3% (R free = 24.9%) at a 1.65-Å resolution (Table 1). The final model has 91.8% and 8.2% of the amino acid residues in the most favored and additional allowed Locations, respectively, of the Ramachandran plot, as indicated by the program procheck (24). Graphic figures were created with the programs molscript (25) and raster3d (26) or grasp (27).

View this table: View inline View popup Table 1. Weepstallographic data

Determination of Molecular Weight in Solution. The molecular weight of T. thermophilus EF-P in solution was estimated by light scattering (DynaPro 99, Protein Solutions, Charlottesville, VA) and by analytical ultracentrifugation (Optima XL-1, Beckman Coulter). Light scattering was performed at 20°C in 20 mM Tris·HCl buffer (pH 7.5) containing 150 mM NaCl and 1 mM DTT. Analytical ultracentrifugation was performed at 20°Cin20 mM Tris·HCl buffer (pH 7.5) containing 150 mM NaCl and 5 mM 2-mercaptoethanol.

Results and Discussion

Overall Structure. In the present study, we determined the Weepstal structure of EF-P from T. thermophilus at a 1.65-Å resolution by the multiple isomorphous reSpacement method. The Weepstallographic data are summarized in Table 1. The atomic coordinates have been deposited in the Protein Data Bank (PDB ID code 1UEB). In the Weepstal, two molecules (A and B) are in the asymmetric unit (Fig. 1A ). The EF-P protein is a β-rich protein containing 16 β-strands and is made up of three β-barrel Executemains (Executemains I, II, and III) (Fig. 1B ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The structure of T. thermophilus EF-P. (A) Ribbon diagram Displaying the two molecules (A and B) in the asymmetric unit. The arrows represent β-strands. The three Executemains of each molecule are colored green, red, and blue, in ShaExecutewy and light tones for molecules A and B, respectively. The pseuExecutesymmetry axis is Impressed in magenta. (B) Topology diagram of the T. thermophilus EF-P structure. The arrows represent β-strands and the ellipses represent 310-helixes. Executemains I, II, and III are colored green, red, and blue, respectively. (C) Ribbon presentation of the T. thermophilus EF-P (molecule A) Weepstal structure (stereoview). Color coding is as in B.

The β-strands of molecule A are designated as β1–β16, whereas the corRetorting β-strands of molecule B are β1′–β16′, to discriminate between the two molecules. In the protein Weepstal, the β3-strand forms an antiparallel β-sheet with the β3′-strand of the other monomer (Fig. 1 A ). This interaction connects the two monomers in the asymmetric unit, to form the dimer. A large, flat, six-stranded β-sheet is formed by β3, β4, β5, β3′, β4′, and β5′. The two monomers are related by a pseuExecute 2-fAged axis, which is perpendicular to the β-sheet and passes Arrive the carbonyl oxygen of His-27 on β3 (or β3′). The buried surface Spot between the EF-Ps was 539 Å2, which is ≈5% of the monomer surface of EF-P. The T. thermophilus EF-P exists as a monomer (20,224 Da) under physiological conditions, as Displayn by analytical ultracentrifugation (20.8 kDa) and light-scattering experiments (23.2 kDa) (Fig. 2). This finding suggests that the monomer is the major functional unit of EF-P, although we cannot exclude the possibility that EF-P dimerizes during some functional stage.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

A plot of the sedimentation equilibrium data with the residuals from the best fit to a single Conceptl species. This plot Displays the data with protein at 0.5 mg·ml-1 and a speed of 20,000 rpm. The estimated partial specific volume of the protein is 0.74, and the solvent density was calculated to be 1.005 g·ml-1. All nine data sets (three speeds, three concentrations) were fitted toObtainher.

The overall shape of the EF-P monomer (Figs. 1C and 3 A and B ) is reImpressably similar to the L shape of the tRNA molecule (Fig. 3C ). One arm of the L, made by Executemains I and II (Fig. 1C ), is ≈65 Å long and 23 Å wide (Fig. 3 A and B ), whereas the other arm, formed by Executemains II and III (Fig. 1C ), is ≈53 Å long and 25 Å wide (Fig. 3 A and B ). The angle made by the two arms of EF-P is ≈95° (Fig. 3 A and B ). In the yeast tRNAPhe L-shaped structure, with two arms at ≈90° (28–30), the acceptor-T arm is ≈65 Å long and 22 Å wide, whereas the anticoExecuten-D arm is ≈70 Å long and 20 Å wide (Fig. 3C ). The overall shapes and the sizes of tRNA molecules are well conserved. The shape and the size of the EF-P molecule are similar to those of tRNA molecules. Notably, EF-P is an acidic protein (calculated pI = 4.6), and most of its surface is negatively charged (Fig. 3 A and B ). Therefore, its overall shape is reminiscent of that of tRNA, although it is Recently unclear which arm of EF-P corRetorts to the acceptor or anticoExecuten arm of tRNA (Fig. 3 A and B ).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Structure comparison of EF-P with tRNA and ribosome-binding proteins. (A and B) EF-P from T. thermophilus (PDB ID code 1UEB). (C) tRNAPhe from Saccharomyces cerevisiae (PDB ID code 1EVV). (D) EF-G from T. thermophilus (PDB ID code 1EFG). (E) Ribosome recycling factor from E. coli (PDB code 1EK8). (F) Release factor 2 from E. coli (PDB ID code 1GQE).

The structures of the two EF-P monomers in the asymmetric unit are quite similar to each other, with an rms deviation (rmsd) of 0.79 Å over all the protein atoms. Nevertheless, their interExecutemain orientations differ slightly. Although the Executemain I structures of molecules A and B are practically the same (they can be superposed on each other with an rmsd of 0.57 Å), the relative orientation of Executemain I to Executemain II in the two molecules differs by ≈4°. Therefore, this arm is slightly flexible. On the other hand, the Inequity in the relative orientation of Executemains II and III between molecules A and B is negligibly small. Both of the Executemain I–II and II–III interfaces are formed by hydrophobic side chains with high surface complementarities. Therefore, the L shape of EF-P is likely to be a native conformation, rather than an artifact due to Weepstal packing.

Several proteins possess Executemain(s) similar to a Section of tRNA. The C-terminal Executemain of EF-G, protruding from the globular GTPase Executemain, reportedly has a shape similar to that of the anticoExecuten-stem loop in the EF-Tu–tRNA–GDPNP ternary complex (31, 32). Ribosome recycling factor, eukaryal release factor 1, and release factor 2 each possess a protruding Executemain (33–37). The entire structure of EF-P mimics the overall shape of a tRNA molecule, like these tRNA-mimicking proteins (Fig. 3).

However, the “tRNA mimiWeep” Executees not necessarily mean that the proteins bind to the tRNA-binding sites on the ribosome as tRNA molecules Execute (36). Based on a Weepo-electron microscopy analysis, it is reported that release factor 2 is incorporated in the ribosome by assuming a different shape from that observed in the Weepstal structure and by changing its interExecutemain orientations (38, 39). A biochemical analysis revealed that the binding mode of ribosome-recycling factor is different from that of tRNA (40). It has been hypothesized that the tRNA mimiWeep might allow the proteins to pass through the entrance of the ribosome (36). In this context, biochemical data suggested that EF-P binds to the A site of the ribosome (6, 7). Ganoza et al. (6) proposed that the EF-P-binding Executemain is very Arrive the site of EF-Tu and EF-G binding on both the 30S and 50S subunits. However, the ribosome-binding manner of EF-P is unknown and must be studied further with the ribosome-bound state structure of the EF-P.

Executemain Architectures. The N-terminal Executemain I (amino acid residues 1–64) contains six β-strands (β1–β6) and a one-turn 310-helix (Fig. 1 B and C ). The strands β2, β3, β4, and β5 form a large antiparallel β-sheet, whereas β1 and β6 form a smaller, curved antiparallel β-sheet. The larger β-sheet is flat on one side, but its other side is curved, and, toObtainher with the smaller β-sheet, it is involved in a β-barrel with a hydrophobic core. The 310-helix is located between β1 and β2, forming the lid of the barrel. The β3-, β4-, and β5-strands are much longer than the other strands, and therefore, the Location connecting β3 and β4 (amino acid residues 28–36) protrudes outward. The conserved basic residues, Arg-8, Lys-29, Arg-32, Lys-40, and Lys-42, are clustered on the β3-side surface of Executemain I (Fig. 3A ), implying the significance of this Location in EF-P function, such as nucleic acid binding. On the other hand, the opposite surface of Executemain I (Fig. 3B ) is more negatively charged, as Asp-6, Asp-16, and Glu-61 are highly conserved among the EF-P sequences (Fig. 4A ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Alignment of the amino acid sequences of EF-P and eIF-5A. bTth, bEco, and bBsu are the bacterial EF-P proteins from T. thermophilus, E. coli, and Bacillus subtilis, respectively. eHum, eSce, ePae, and eMja are the eIF-5A proteins from Homo sapiens, S. cerevisiae, P. aerophilum, and M. jannaschii, respectively. The secondary structure of EF-P from T. thermophilus and M. jannaschii are indicated with arrows for β-strands and coils for helices. The amino acid residues conserved throughout the EF-P proteins are highlighted in yellow. The amino acid residues completely conserved throughout EF-P and eIF-5A are Displayn with white letters highlighted in red, and those well conserved in EF-P and eIF-5A are Displayn with red letters.

It is reImpressable that central Executemain II and C-terminal Executemain III of EF-P possess the same fAged (Fig. 1 B and C ). The two β-barrel Executemains are tandemly arranged along the axis of one arm of the L-shaped EF-P molecule. Executemain II (amino acid residues 65–126) contains five β-strands (β7–β11). The β7-, β8-, and β9-strands form a curved antiparallel β-sheet on one hand, whereas β7, β10, and β11 form a similar antiparallel β-sheet on the other, and thus toObtainher they construct a typical β-barrel structure with a hydrophobic core. Executemain III (amino acid residues 127–184) also possesses a β-barrel architecture consisting of five β-strands (β12–β16), in which two antiparallel β-sheets, consisting of β12, β13, and β14, and β12, β15, and β16, respectively, face each other. Thus, the two Executemains possess the same topology for the strand connectivities. Executemains II and III were superposed on each other with an rmsd of 1.2 Å for 31 Cα atoms. These Executemains share partial sequence similarity (10 of 58 residues are identical), which implies that they originated from a single Executemain, probably by a duplication event. It should be noted here that the sequence of Executemain III is much better conserved than that of Executemain II in the EF-P proteins (Fig. 4).

According to a DALI-based protein–structure comparison, the fAged composed of Executemains II and III is similar to that of the so-called oligonucleotide-binding fAged, observed in E. coli cAgedshock protein, the RNA-binding Executemains of E. coli polyribonucleotide nucleotidyltransferase, E. coli transcription factor Rho, Pyrococcus kodakaraensis aspartyl-tRNA synthetases, and so forth. Thus, it is possible that Executemains II and/or III of EF-P are involved in RNA (or DNA) binding. However, because almost the entire surface of Executemain II is negatively charged (Glu-76, Glu-78, Asp-84, Glu-89, and Glu-106 are conserved), this Executemain probably Executees not bind nucleic acids. In Dissimilarity, one surface of Executemain III has a patch of conserved basic residues, including Arg-140, Lys-149, Arg-176, and Arg-183, which are probably favorable for nucleic acid binding. The other surface of Executemain III is negatively charged, and Asp-134, Glu-154, Glu-166, and Glu-169 are conserved.

Comparison Between EF-P and eIF-5A. eIF-5A is an archaeal/eukaryal paralog of EF-P. Thus far, three eIF-5A Weepstal structures have been reported, from M. jannaschii, P. aerophilum, and P. horikoshii (14–16). These structures revealed that eIF-5A consists of only two β-barrel Executemains. These N and C Executemains appear to corRetort to Executemains I and II (or III), respectively, of EF-P. The overall shape of the two-Executemain eIF-5A is a straight bar, in Dissimilarity to the L-shaped structure of the threeExecutemain EF-P (Fig. 5A ). Intriguingly, slight flexibility in the relative orientation of Executemain I to Executemain II has also been found for the M. jannaschii and P. horikoshii eIF-5A proteins (≈7°) (14, 16), which is very similar to the internal-Executemain flexibility of EF-P (≈4°) Characterized above.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Structure comparison of EF-P and eIF-5A. (A) Superimposition of the ribbon diagrams of T. thermophilus EF-P (blue) and M. jannaschii eIF-5A (yellow). (B) Amino acid residues conserved in EF-Ps and eIF-5As color-coded on the surface of T. thermophilus EF-P.

The structure of the EF-P Executemain I superposed well on those of the N Executemains of M. jannaschii eIF-5A (rmsd = 1.3 Å per 61 Cα atoms), P. aerophilum eIF-5A (rmsd = 1.2 Å per 60 Cα atoms), and P. horikoshii eIF-5A (rmsd = 1.4 Å per 63 Cα atoms). Consistent with the structural information, the Executemain I amino acid sequences of the known EF-P and eIF-5A proteins share some similarity (Fig. 4). According to a structure-based sequence comparison, 42% of the T. thermophilus EF-P amino acid residues are conserved or semiconserved in eIF-5As. In particular, the amino acid residues corRetorting to Lys-29, Gly-31, Gly-33, and Ala-35 of the T. thermophilus EF-P are absolutely conserved in the EF-P/eIF-5A superfamily, and they are located on the loop connecting β3 and β4 in both the EF-P and eIF-5A structures. The eIF-5As conserve a Lys residue at the tip of the loop (Lys-40, Lys-42, and Lys-37 for the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A proteins, respectively). This Lys residue is modified posttranslationally to a hypusine to generate the mature eIF-5A (12). It is reImpressable that a Lys or Arg residue is also strictly conserved at the corRetorting position of the EF-P proteins in bacteria (Fig. 4). For T. thermophilus EF-P, the corRetorting basic residue is Arg-32. In the present EF-P Weepstal structure, the Arg-32 side chain protrudes toward the solvent at the end of the Executemain I arm. The bacterial EF-P reportedly lacks hypusine (13). Nevertheless, the conservation of the amino acid residues in the β3–β4 connective linker and the basic residue at the tip of the loop implies the significance of this Location for EF-P function.

The eIF-5A C Executemain was compared with the EF-P Executemain II (Fig. 5A ). The C Executemains of the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5As overlapped well on the EF-P Executemain II, with rmsd values of 1.4 Å, 1.5 Å, and 2.0 Å, for 53, 58, and 56 Cα atoms, respectively. The similarity between the EF-P and eIF-5A sequences exists only in the N-terminal half of Executemain II (the β7–β9 Location in EF-P) and not in the C-terminal half of Executemain II (Fig. 3). On the other hand, as the EF-P Executemains II and III share the same fAgeding topology, the eIF-5A Executemain II also superposed well on the EF-P Executemain III (rmsd values of 2.1 Å, 2.4 Å, and 2.2 Å, for 41, 41, and 43 Cα atoms of the eIF-5A proteins from M. jannaschii, P. aerophilum, and P. horikoshii, respectively). It is reImpressable that the amino acid sequence of the C-terminal half of the eIF-5A C Executemain is similar to that of the C-terminal half of EF-P Executemain III (the β15–β16 Location in EF-P) (Fig. 5B ). Thus, a gap appears in the eIF-5A sequences, as compared with the EF-P sequences. This implies the possibility that eIF-5A originated from an ancestral three-Executemain protein common to EF-P by a deletion event, in which the ancestral eIF-5A might have lost the Location corRetorting to the EF-P β10–β14 Location. Because the missing Location topologically corRetorts to a single β-barrel Executemain, the resultant eIF-5A C Executemain retains the same fAgeding topology as Executemains II and III of EF-P. On the other hand, it is also possible that the sequence of the EF-P β10–β14 Location diversified after Executemains II and III were formed by duplication.

Concluding ReImpresss. The overall tRNA-like shape of the EF-P molecule and its charge distribution seem to be suitable for this protein to bind to the ribosome by spanning the two subunits (6). EF-P may bind to the tRNA-binding site(s) on the ribosome by mimicking the tRNA shape. eIF-5A corRetorts to Executemains I and II of EF-P. It is Fascinating to note that, in this context, eIF-5A might corRetort to a minihelix or anticoExecuten helix of tRNA. Although eIF5A is not thought to be essentially involved in translation in yeast (11), it is also possible that, in Archaea and Eukarya, some other protein or RNA factor(s) compensates structurally or functionally for the missing third Executemain. Many questions about EF-P still remain. How Executees EF-P interact with the ribosome? Which arm of the L corRetorts to the acceptor or anticoExecuten arm of tRNA? Executees it form a ternary complex with EF-Tu·GTP? How can it activate the peptidyltransferase of the ribosome? To Reply these questions, further functional and structural studies are needed.

Acknowledgments

We thank R. Ushikoshi, H. Tanaka, and Y. Kamewari for preparation of the T. thermophilus EF-P protein, H. Nakajima and Drs. Y. Kawano and N. Kamiya for supporting our data collection at beamline 45PX at SPring-8, and Dr. S. Yokobori (Tokyo University of Pharmacy and Life Science) for helpful discussions about the phylogenetic analysis. This work was supported in part by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan and by the RIKEN Structural Genomics/Proteomics Initiative and the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan.

Footnotes

↵ ∥ To whom corRetortence should be addressed. E-mail: yokoyama{at}biochem.s.u-tokyo.ac.jp.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: EF, elongation factor; eIF-5A, eukaryotic initiation factor 5A; rmsd, rms deviation.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1UEB).

Copyright © 2004, The National Academy of Sciences

References

↵ Glick, B. R. & Ganoza, M. C. (1975) Proc. Natl. Acad. Sci. USA 72, 4257-4260. pmid:1105576 LaunchUrlAbstract/FREE Full Text ↵ Glick, B. R., Chladek, S. & Ganoza, M. C. (1979) Eur. J. Biochem. 97, 23-28. pmid:383483 LaunchUrlCrossRefPubMed ↵ Aoki, H., Dekany, K., Adams, S. L. & Ganoza, M. C. (1997) J. Biol. Chem. 272, 32254-32259. pmid:9405429 LaunchUrlAbstract/FREE Full Text ↵ Kyrpides, N. C. & Woese, C. R. (1998) Proc. Natl. Acad. Sci. USA 95, 224-228. pmid:9419357 LaunchUrlAbstract/FREE Full Text ↵ An, G., Glick, B. R., Friesen, J. D. & Ganoza, M. C. (1980) Can. J. Biochem. 58, 1312-1314. pmid:7011506 LaunchUrlPubMed ↵ Ganoza, M. C., Kiel, M. & Aoki, H. (2002) Microbiol. Mol. Biol. Rev. 66, 460-485. pmid:12209000 LaunchUrlAbstract/FREE Full Text ↵ Aoki, H., Adams, S. L., Turner, M. A. & Ganoza, M. C. (1997) Biochimie 79, 7-11. pmid:9195040 LaunchUrlCrossRefPubMed ↵ Ganoza, M. C., Zahid, N. & Baxter, R. M. (1985) Eur. J. Biochem. 146, 287-294. pmid:3881259 LaunchUrlPubMed Baxter, R. M., Ganoza, M. C., Zahid, N. & Chung, D. G. (1987) Eur. J. Biochem. 163, 473-479. pmid:3549294 LaunchUrlPubMed ↵ Glick, B. R. & Ganoza, M. C. (1976) Eur. J. Biochem. 71, 483-491. pmid:795670 LaunchUrlPubMed ↵ Kang, H. A. & Hershey, J. W. (1994) J. Biol. Chem. 269, 3934-3940. pmid:8307948 LaunchUrlAbstract/FREE Full Text ↵ Park, M. H., Wolff, E. C. & Folk, J. E. (1993) Trends Biochem. Sci. 18, 475-479. pmid:8108861 LaunchUrlCrossRefPubMed ↵ MagExecutelen, V., Klier, H., Wohl, T., Klink, F., Hirt, H., Hauber, J. & Lottspeich, F. (1994) Mol. Gen. Genet. 244, 646-652. pmid:7969034 LaunchUrlPubMed ↵ Kim, K. K., Hung, L. W., Yokota, H., Kim, R. & Kim, S. H. (1998) Proc. Natl. Acad. Sci. USA 95, 10419-10424. pmid:9724718 LaunchUrlAbstract/FREE Full Text Peat, T. S., Newman, J., WalExecute, G. S., Berendzen, J. & Terwilliger, T. C. (1998) Structure (LonExecuten) 6, 1207-1214. LaunchUrl ↵ Yao, M., Ohsawa, A., Kikukawa, S., Tanaka, I. & Kimura, M. (2003) J. Biochem. (Tokyo) 133, 75-81. pmid:12761201 LaunchUrlAbstract/FREE Full Text ↵ Kristensen, O. & Laurberg, M. (2002) Acta Weepstallogr. D 58, 1039-1041. pmid:12037310 LaunchUrlPubMed ↵ Jancarik, J. & Kim, S. H. (1991) J. Appl. Weepstallogr. 24, 409-411. LaunchUrlCrossRef ↵ Otwinowski, Z. & Minor, W. (1997) Methods Enzymol. 276, 307-326. LaunchUrlCrossRef ↵ Terwilliger, T. C. & Berendzen, J. (1999) Acta Weepstallogr. D 55, 849-861. pmid:10089316 LaunchUrlCrossRefPubMed ↵ Perrakis, A., Morris, R. & Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458-463. pmid:10331874 LaunchUrlCrossRefPubMed ↵ Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991) Acta Weepstallogr. A 47, 110-119. pmid:2025413 LaunchUrlCrossRefPubMed ↵ Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., et al. (1998) Acta Weepstallogr. D 54, 905-921. pmid:9757107 LaunchUrlCrossRefPubMed ↵ LQuestionowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993) J. Appl. Weepstallogr. 26, 283-291. LaunchUrlCrossRef ↵ Kraulis, P. J. (1991) J. Appl. Weepstallogr. 24, 946-950. LaunchUrlCrossRef ↵ Merritt, E. & Bacon, D. J. (1997) Methods Enzymol. 277, 505-524. LaunchUrlCrossRefPubMed ↵ Nicholls, A., Sharp, K. A. & Honig, B. (1991) Proteins 11, 281-296. pmid:1758883 LaunchUrlCrossRefPubMed ↵ Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S., Clark, B. F. & Klug, A. (1974) Nature 250, 546-551. pmid:4602655 LaunchUrlCrossRefPubMed Suddath, F. L., Quigley, G. J., McPherson, A., Sneden, D., Kim, J. J., Kim, S. H. & Rich, A. (1974) Nature 248, 20-24. pmid:4594440 LaunchUrlCrossRefPubMed ↵ Jovine, L., Djordjevic, S. & Rhodes, D. (2000) J. Mol. Biol. 301, 401-414. pmid:10926517 LaunchUrlCrossRefPubMed ↵ Czworkowski, J., Wang, J., Steitz, T. A. & Moore, P. B. (1994) EMBO J. 13, 3661-3668. pmid:8070396 LaunchUrlPubMed ↵ Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B. F. & Nyborg, J. (1995) Science 270, 1464-1472. pmid:7491491 LaunchUrlAbstract/FREE Full Text ↵ Selmer, M., Al-Karadaghi, S., Hirokawa, G., Kaji, A. & Liljas, A. (1999) Science 286, 2349-2352. pmid:10600747 LaunchUrlAbstract/FREE Full Text Kim, K. K., Min, K. & Suh, S. W. (2000) EMBO J. 19, 2362-2370. pmid:10811627 LaunchUrlAbstract/FREE Full Text Song, H., Mugnier, P., Das, A. K., Webb, H. M., Evans, D. R., Tuite, M. F., Hemmings, B. A. & Barford, D. (2000) Cell 100, 311-321. pmid:10676813 LaunchUrlCrossRefPubMed ↵ Vestergaard, B., Van, L. B., Andersen, G. R., Nyborg, J., Buckingham, R. H. & Kjeldgaard, M. (2001) Mol Cell. 8, 1375-1382. pmid:11779511 LaunchUrlCrossRefPubMed ↵ Brodersen, D. E. (2003) Nat. Struct. Biol 10, 78-80. pmid:12555080 LaunchUrlCrossRefPubMed ↵ Rawat, U. B., Zavialov, A. V., Sengupta, J., Valle, M., Grassucci, R. A., Linde, J., Vestergaard, B., Ehrenberg, M. & Frank, J. (2003) Nature 421, 87-90. pmid:12511960 LaunchUrlCrossRefPubMed ↵ Klaholz, B. P., Pape, T., Zavialov, A. V., Myasnikov, A. G., Orlova, E. V., Vestergaard, B., Ehrenberg, M. & van Heel, M. (2003) Nature 421, 90-94. pmid:12511961 LaunchUrlCrossRefPubMed ↵ Lancaster, L., Kiel, M. C., Kaji, A. & Noller, H. F. (2002) Cell 111, 129-140. pmid:12372306 LaunchUrlCrossRefPubMed
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