Mutagenesis of Arg335 in Bovine Mitochondrial Elongation Factor Tu ...

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EF-Tu is composed of three domains.2-4 Domain I is the guanine nucleotide binding .... 2.5 mM phosphoenol pyruvate, 0.5 units pyruvate kinase, 0.5 mM GTP.
[RNA Biology 1:2, 95-102; July/August 2004]; ©2004 Landes Bioscience

Mutagenesis of Arg335 in Bovine Mitochondrial Elongation Factor Tu and the Corresponding Residue in the Escherichia coli Factor Affects Interactions with Mitochondrial Aminoacyl-tRNAs Research Paper

Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=1034

KEY WORDS mitochondria, bacteria, protein synthesis, elongation, elongation factor Tu mutagenesis, tRNA

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ACKNOWLEDGEMENTS

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Received 04/24/04; Accepted 06/16/04

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*Correspondence to: Linda L. Spremulli; Department of Chemistry; Campus Box 3290; University of North Carolina; Chapel Hill, North Carolina 27599-3290 USA; Tel.: 919.966.1567; Fax: 919.966.3675; Email: [email protected]

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address: Department of Molecular Biophysics and Biochemistry; P.O. Box 208114; 266 Whitney Avenue; Yale University; New Haven, Connecticut 065208114 USA †Current

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of the ribosome. Mammalian mitochondrial EF-Tu (EF-Tumt) carries out this activity using aa-tRNAs that lack many of the invariant or semi-invariant residues that stabilize the 3-dimensional structures of canonical tRNAs. The primary sequence of EF-Tu is highly conserved. However, several residues involved in aa-tRNA binding are not conserved between the mitochondrial and bacterial factors. One such residue, located at position 287 in Escherichia coli EF-Tu, is adjacent to the 5’ end of the aa-tRNA and is acidic in all prokaryotic factors but is basic in EF-Tumt. Site-directed mutagenesis of this residue (Glu287) in E. coli EF-Tu and complementary mutagenesis of the corresponding Arg335 in EF-Tumt was performed to create E. coli EF-Tu E287R and EF-Tumt R335E respectively. EF-Tumt R335E has a reduced activity in ternary complex formation and A-site binding with mitochondrial Phe-tRNA.Phe In contrast, E. coli EF-Tu E287R is more active that the wild-type factor in forming ternary complexes with mitochondrial Phe-tRNA,Phe and the variant promotes the binding of mitochondrial aa-tRNA to the ribosome more effectively than does the wild-type factor. Both EF-Tumt R335E and E. coli EF-Tu E287R have activities comparable to the corresponding wild-type factors in assays using E. coli Phe-tRNA.Phe These data suggest that the residue at position 287 plays an important role in the binding and EF-Tu-mediated delivery of mitochondrial aa-tRNAs to the A-site of the ribosome.

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Department of Chemistry; University of North Carolina; Chapel Hill, North Carolina USA

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ABSTRACT

Senyene Eyo Hunter† Linda L. Spremulli*

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This work has been supported in part by funds provided by the National Institutes of Health (Grant GM32734 to L.L.S.) and by the National Institutes of Health NRSA (1F31GM64007 to S.E.H.).

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During the elongation phase of protein synthesis, the active, EF-Tu•GTP complex binds aminoacyl-tRNA (aa-tRNA) forming a ternary complex through which the aa-tRNA is delivered to the A-site of the ribosome. Following codon recognition, GTP is hydrolyzed and the inactive EF-Tu•GDP complex dissociates from the ribosome. The guanine nucleotide exchange factor, elongation factor Ts (EF-Ts), then serves to release GDP from EF-Tu forming an EF-Tu•EF-Ts complex. EF-Ts is replaced by GTP and the active EF-Tu•GTP complex binds another aa-tRNA allowing repetition of the cycle.1 EF-Tu is composed of three domains.2-4 Domain I is the guanine nucleotide binding domain. Domains I and III interact with EF-Ts while all three domains contact the aa-tRNA in the ternary complex.2,3,5 Mammalian mitochondria contain a protein synthesizing system that has significant similarities to the prokaryotic translational system. The primary sequence of EF-Tumt is 56% identical to that of E. coli EF-Tu. The crystal structure of the EF-Tumt•GDP complex has been determined at 1.94 Å resolution.6 The three-dimensional structure of the mitochondrial factor is very similar to that observed for prokaryotic EF-Tu. One striking feature of mitochondrial protein synthesis is the structure of the tRNAs present in this organelle.7 The primary sequences of the classical tRNAs that function in prokaryotic and eukaryotic cytoplasmic protein synthesis contain conserved or semi-conserved residues that enable the tRNAs to fold into stable “L” shaped structures. These canonical tRNAs also contain a high G-C content in their stem regions increasing their stability.8 In contrast, mammalian mitochondrial tRNAs lack a number of these invariant and semi-invariant residues; they have higher A-U pairing in their stem regions and they are often significantly shorter than other tRNAs with as few as 59 nucleotides compared to 75 or more in prokaryotic tRNAs.6,8 The atypical mitochondrial tRNAs are less stable than canonical tRNAs and display lower melting temperatures compared to other tRNAs.9 Both E. coli EF-Tu and EF-Tumt are able to form ternary complexes with mitochondrial aa-tRNAs. However, compared to EF-Tumt, E. coli EF-Tu displays a reduced ability to deliver these aa-tRNAs to the A-site of the ribosome.10 The lack of many of the normally conserved features in mitochondrial aa-tRNAs may affect their tertiary structures and

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reduce the ability of E. coli EF-Tu to position them effectively in the A-site of the ribosome. Most of the residues that comprise the aa-tRNA binding site in EF-Tu are completely conserved or semi-conserved among the factors from various species (Fig. 1). This observation suggests that, while these residues may be in direct contact with the aa-tRNA, they do not contribute to the differences in the abilities of E. coli EF-Tu and EF-Tumt to promote mitochondrial aa-tRNA binding to ribosomes. However, residue 287 is a striking exception to the conservation of residues making contact with the aa-tRNA or in close proximity to its binding site on EF-Tu (Fig. 1). This residue is a Glu (or occasionally a Gln) in the prokaryotic factors but is a basic residue, Lys or Arg, in the mitochondrial factors from a variety of organisms. This residue is Arg335 in bovine Figure 1. Primary sequence alignment of Domain II of EF-Tu. The arrow indicates the residue of EF-Tumt. interest. The “*” symbols specify residues involved in binding the 3’ acceptor stem region of the To help elucidate the properties of EF-Tumt aa-tRNA. The “†” symbols identify residues involved in binding the 5’ end of the aa-tRNA. The that allow it to interact with and deliver mito- accession numbers for each factor are as follows: Escherichia coli EF-Tu (E. coli)—P02290; chondrial aa-tRNA to ribosomes, Arg335 in Bacillus subtilis EF-Tu (B. subtilis)—P33166; Rickettsia prowazekii EF-Tu (R. prowazekii)— EF-Tumt was converted to a Glu to create EF-Tumt AJ235272; Thermus aquaticus EF-Tu (T. aquaticus)—S29293; Saccharomyces cerevisiae EF-Tumt R335E. The complementary mutagenesis of the (S. cerevisiae)—K00428; Mus musculus EF-Tumt (M. musculus)—XM_133763; Bos bovis EF-Tumt (B. bovis)—P49410; Homo sapiens EF-Tumt (H. sapien)—BC010041. Glu287 of E. coli EF-Tu was performed, generating E. coli EF-Tu E287R. The effects of these mutations on the function of prokaryotic and mitochondrial EF-Tu were Plasmids coding for the expression of either E. coli EF-Tu E287R or EF-Tumt R335E were transformed into E. coli BL21(DE3) cells. Cell characterized in detail.

MATERIALS AND METHODS

Materials. All enzymes used for cloning were obtained from New England Biolabs except Pfu high-fidelity polymerase which was acquired from Stratagene. Radioactive materials were purchased from Perkin Elmer Life Sciences Inc. E. coli tRNA was obtained from Boehringer Mannheim. [14C]Phe-tRNAPhe and [35S]Cys-tRNACys were prepared as described.11 E. coli ribosomes and elongation factor G were prepared from E. coli W.10,12 Mitochondrial EF-G 12,13 and crude mitochondrial ribosomes14 were purified from bovine liver. Purified mitochondrial ribosomes were prepared as described.15 E. coli and mitochondrial EF-Ts were expressed in E. coli as His-tagged variants and purified on Ni-NTA resin.12,16 Bovine mitochondrial tRNA was isolated from bovine mitoplasts (mitochondria from which the outer membrane has been removed) using the Qiagen RNA/DNA Maxi Kit. Mitochondrial tRNAPhe was aminoacylated with [14C]Phe using human mitochondrial Phe-tRNAPhe synthetase.17 Superase•In RNase inhibitor was purchased from Ambion. Nitrocellulose membrane filters were type HA (Millipore Corporation). Cloning and Expression of E. coli EF-Tu E287R and EF-Tumt R335E. The gene coding for E. coli EF-Tu E287R was produced using the C-terminally His-tag variant of wild-type E. coli EF-Tu DNA cloned into pET-24c(+) as a template as described by the Stratagene QuikChange Site Directed Mutagenesis protocol.16 The forward and reverse synthetic oligonucleotides used to introduce the mutations were 5’-gtgaagaaatccgtcgtggtcaggtactggctaagccg-3’ and 5’-cctgaccacgacggatttcttcacgtttgatac-3’ respectively. The R335E variant of EF-Tumt was prepared using similar procedures except the template used was the C-terminally His-tag coding region of EF-Tumt inserted into pET-24c(+)18 and the forward and reverse oligonucleotides were 5’-gggaggacctggaacgtggcctggtcatggcc-3’ and 5’-accaggcaacgttccaggtcctcccgcttcaagcctc-3’ respectively. Mutated plasmids were transformed into E. coli DH5α cells and the sequences were verified at the Automated DNA Sequencing Facility at the University of North Carolina at Chapel Hill.

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extracts were prepared and wild-type or variant EF-Tu were purified as described previously19 except that buffers contained 10 or 50 µM GDP or 50 µM GTP as indicated. In Vitro Translation. The abilities of the variant and wild-type EF-Tu to carry out poly(U)-directed polymerization of [14C]Phe were determined as described.10,12,13,20 Input values of EF-Tu were corrected for the percentage of molecules active in ternary complex formation (see below). The amount of [14C]Phe polymerized was corrected for the blank in each assay (the amount of radioactivity obtained in a minus EF-Tu control, approximately 0.5 pmol). In vitro translation assays using mitochondrial ribosomes were carried out as described.20 Ternary Complex Formation. RNase Protection Assay. Ternary complex formation was assayed by examining the ability of EF-Tu to protect E. coli [35S]Cys-tRNACys, E. coli [14C]Phe-tRNAPhe or mitochondrial [14C]Phe-tRNAPhe from hydrolysis by RNase A.12 The indicated amount of wild-type or variant EF-Tumt prepared in 50 µM GDP was incubated with 2.5 mM phosphoenol pyruvate, 0.5 units pyruvate kinase, 0.5 mM GTP and the indicated amount of aa-tRNA in a 50 µL reaction mixture containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 68 mM KCl and 6.7 mM MgCl2. After incubation at 0˚C for 15 min, 10 µg RNase A was added to digest the free aa-tRNA. Following an additional 30 s incubation at 0˚C the reaction was terminated by the addition of cold 5% trichloroacetic acid. The precipitate was collected on nitrocellulose filters following a 10 min incubation on ice and the amount of E. coli [14C]Phe-tRNAPhe, [35S]Cys-tRNACys or mitochondrial [14C]Phe-tRNAPhe remaining was quantified using a liquid scintillation counter. The quantities of radiolabeled aa-tRNAs obtained are corrected for the amount of radiolabel precipitated in a minus EF-Tu control (approximately about 0.5 pmol for E. coli [35S]Cys-tRNACys and 0.2 pmol for [14C]-labeled Phe-tRNAs).12,13 To allow the conversion of GDP to GTP in the bacterial EF-Tu preparations, ternary complex formation with wild-type and variant E. coli EF-Tu was carried out as described above except that prior to the 15 min incubation at 0˚C, EF-Tu (0.07–0.16 µM) prepared in 50 µM GTP was incubated for 2–2.5 h in the reaction mixture (40 µL) containing phosphoenol pyruvate and pyruvate kinase. Radiolabeled aa-tRNA (0.1–0.5 µM) was then

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added in 10 µL to achieve the final concentrations listed in the legends. The determination of the percentage of active EF-Tu molecules indicated that the prolonged incubation period did not result in any loss of activity. Nonenzymatic Hydrolysis Protection Assay. The ability of wild-type and variant EF-Tu to protect Phe-tRNAPhe from spontaneous deacylation was monitored as described.21,22 Wild-type and variant EF-Tumt (1.0 µM) containing 10 µM GDP were incubated with either E. coli or mitochondrial [14C]Phe-tRNAPhe (0.40 µM) at 0˚C for 20 min in a 100 µL reaction mixture containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 71 mM KCl, 6.5 mM MgCl2, 2.5 mM phosphoenol pyruvate, 1.0 unit pyruvate kinase and 0.5 mM GTP. The reactions were shifted to 20˚C and aliquots (10 µL) were withdrawn at various times, precipitated in 5% trichloroacetic acid and treated as described above. The half-life of the ternary complex was determined from a plot of ln(xt/x0) versus time where xt is the concentration of EF-Tu•GTP•[14C]Phe-tRNAPhe at time = t and x0 is the concentration of EF-Tu•GTP•[14C]Phe-tRNAPhe at time t = 0. The activity of wild-type and variant E. coli EF-Tu in nonenzymatic protection assays was carried out as described above except that prior to the addition of radiolabeled aa-tRNA, EF-Tu prepared in 10 µM GDP was incubated for 2.5 h in the reaction mixture containing phosphoenol pyruvate and pyruvate kinase to allow the conversion of GDP to GTP. Determining the Percentage of Active Molecules. The percentage of EF-Tu molecules active in forming ternary complexes with GTP and E. coli [14C]Phe-tRNAPhe was determined by a method similar to that above.23 In these assays, a low concentration of EF-Tu (0.2 µM, as determined by Bradford assays) was incubated with increasing concentrations of E. coli [14C]Phe-tRNAPhe (up to 1 µM) under the conditions described above. The maximum amount of ternary complex that can be formed under these conditions is controlled by the amount of active EF-Tu present in the assay. The concentration of EF-Tu active in ternary complex formation is determined directly from this maximum value after correcting for the background (the amount of label precipitated in a minus EF-Tu control at each aa-tRNA concentration—about 0.4 pmol). The percentage of active EF-Tu is the concentration of active molecules determined from the assay divided by the physical amount of EF-Tu present as determined by the Bradford assay. Unless otherwise indicated, input values of EF-Tu used in all assays are corrected for the percentage of EF-Tu molecules active in ternary complex formation. Filter binding assays were used to measure the percentage of variant and wild-type E. coli EF-Tu active in binding GDP.24 Reaction mixtures (120 µL) contained 8.3 to 83 nM EF-Tu (as determined by Bradford assays) and a saturating level (0.4 µM) of [3H]GDP. The percentage of EF-Tu active in binding GDP was determined from the point at which the factor was saturated with GDP after correcting for the background (retention of radiolabeled GDP in a minus EF-Tu control—less than 0.1 pmol). Determining the Dissociation Rate of the EF-Tu•GTP•Phe-tRNAPhe Complex. The dissociation of the ternary complex was monitored as described.22,25 Wild-type and variant E. coli EF-Tu (1.08 µM and 1.02 µM respectively) containing 10 µM GDP were incubated in a 100 µL reaction mixture containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 71 mM KCl, 6.5 mM MgCl2, 2.5 mM phosphoenol pyruvate, 1.0 unit pyruvate kinase and 0.5 mM GTP at 37˚C for 2.5 h to regenerate GTP in the EF-Tu samples. Immediately following this incubation, 10 µL of each sample was removed and the percentage of active EF-Tu molecules was determined as described above to ensure that activity was not lost during the incubation. To the remaining 90 µL reaction mixture, [14C]Phe-tRNAPhe was added to 0.40 µM and the reaction was incubated on ice an additional 20 min to allow ternary complex formation. Following the incubation, RNase A was added to 10 µg/mL. Aliquots (10 µL) were withdrawn at the specified times, precipitated in 5% trichloroacetic acid and treated as described above. A blank (reaction mixture lacking EF-Tu) was carried out for each assay and the background (less than 0.07 pmol) was subtracted from each value. The dissociation rate was determined according to the equation: ln(xt/x0) = -kt where t is the incubation time, xt is the concentration of EF-Tu•GTP•[14C]Phe-tRNAPhe at time = t, x0 is the concentration of EF-Tu•GTP•[14C]Phe-tRNAPhe at time t = 0 and k is the dissociation rate constant.

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A-Site Binding Assays. The ability of wild-type and variant EF-Tu to deliver aa-tRNA to the A-site of the ribosome was assayed as described.10 The reaction mixtures (100 µL) containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 0.1 mM spermine, 80 mM KCl, 6 mM MgCl2, 0.5 mM guanosine 5’-[β, γ(-imido]triphosphate (GDPNP, a nonhydrolyzable analogue of GTP), 0.125 mg/mL poly(U), 50 units Superase•In, 0.38 µM purified E. coli ribosomes, 0.05 µM mitochondrial [14C]Phe-tRNAPhe or 0.1 µM E. coli [14C]Phe-tRNAPhe with 25 µg crude E. coli tRNA and the indicated amount of EF-Tu were incubated at 37˚C for 5 min. The amount of [14C] Phe-tRNAPhe bound to the ribosome was determined using a filter-binding assay and measured with a liquid scintillation counter. All values calculated for A-site binding are corrected for the background in each assay (retention of [14C]Phe-tRNAPhe in a minus EF-Tu control—about 0.8 pmol with E. coli [14C]Phe-tRNAPhe and approximately 0.2 pmol using mitochondrial [14C]Phe-tRNAPhe). The input values of EF-Tu were corrected for the percentage of molecules capable of binding E. coli aa-tRNA. Removal of Excess GDP from EF-Tu and Determination of the Affinity of EF-Tu for GDP. Wild-type E. coli EF-Tu and E. coli EF-Tu E287R were prepared in the presence of 10 µM GDP as described above and a sample (250 µL containing 700 pmol as determined by the percentage of molecules active in GDP binding) was applied to a Sephadex G-50 column (13 x 180 mm, 0.5 mL/min flow rate) equilibrated in Buffer I (40 mM Tris-HCl [pH 7.8], 8.1 mM MgCl2, 130 mM NH4Cl and 1 mM dithiothreitol) at 4˚C.26,27 EF-Tu was eluted from the Sephadex G-50 column as a 1:1 EF-Tu•GDP complex. Fractions (0.1 mL) containing EF-Tu•GDP were pooled and immediately tested for the percentage of EF-Tu active in GDP binding as described above. GDP binding measurements were carried out using the filter binding assay.24 EF-Tu (3–30 nM) was incubated in a reaction mixture (120 µL) containing Buffer I, 0.2 mg/mL bovine serum albumin and 2 nM [3H]GDP (7000 cpm/pmol) for 10 min at 37˚C. The concentration of EF-Tu active in binding GDP was determined in each assay by combining 20–40 nM of EF-Tu with 0.4 µM [3H]GDP (600 cpm/pmol). The specific activities of [3H]GDP were corrected for the amount of GDP present in the EF-Tu preparation, which was determined immediately following purification on the Sephadex G-50 columns. The binding of GDP was corrected for the retention of radiolabeled GDP in a minus EF-Tu control (less than 0.1 pmol). Preparation of Nucleotide-Free EF-Tu and Determination of the Dissociation Rate of the EF-Tu•GDP Complex. Nucleotide-free EF-Tu was prepared and used to determine the dissociation rate of the EF-Tu• GDP complex.27 Wild-type E. coli EF-Tu and E. coli EF-Tu E287R were prepared in the presence of 10 µM GDP as described above. To remove GDP from the EF-Tu preparations, a portion of EF-Tu (600 pmol as determined by the percentage of active molecules in GDP binding) was incubated in 5 mM EDTA at 37˚C for 13 min. The sample was applied to a Sephadex G-50 column (13 x 180 mm, 0.5 mL/min flow rate) equilibrated in Buffer I lacking MgCl2. The fractions containing EF-Tu were pooled and MgCl2 was added to a concentration of 10 mM. [3H]GDP (0.5 µM) was added to the EF-Tu samples and the specific activity was determined for each sample (approximately 12,000 cpm/pmol). The concentration of EF-Tu•[3H]GDP was approximately 0.48 µM (determined by the percentage of active molecules in GDP binding), indicating that the amount of [3H]GDP did not significantly exceed that of EF-Tu. EF-Tu•[3H]GDP was used to determine the dissociation rate constant for the release of GDP from EF-Tu. EF-Tu•[3H]GDP (12.5 nM) was mixed in a 2 mL reaction containing 0.02 mM GDP and 0.2 mg/mL bovine serum albumin in Buffer I. Aliquots (200 µL) were assayed for the concentration of EF-Tu•[3H]GDP remaining after the indicated times using a filter binding assay. The dissociation rate was determined according to the equation: ln(xt/x0) = -kt where t is the incubation time, xt is the concentration of EF-Tu•[3H]GDP at time = t, x0 is the concentration of EF-Tu•[3H]GDP at time t = 0 and k is the dissociation rate constant. Preparation of Nucleotide-Free EF-Tu and Monitoring EF-Tu•GTP Complex Formation. Nucleotide-free wild-type E. coli EF-Tu and E. coli EF-Tu E287R were prepared with a 2% charcoal solution as described28 except reactions were incubated on ice for 7 min and centrifuged for 3 min

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to minimize the loss of activity of the factors. Subjecting a preformed EF-Tu•[3H]GDP complex to this charcoal method indicated that more than 98% of the EF-Tu was nucleotide free after treatment. The percentage of EF-Tu recovered afer the charcoal treatment (generally greater than 90%) was determined by testing the ability of the charcoal-treated EF-Tu to bind [3H]GDP. The binding of wild-type E. coli EF-Tu and its E287R variant to [γ-32P]GTP was carried out as described above except reaction mixtures (120 µL) contained 6.9 µM [γ-32P]GTP (20,000 cpm/ pmol), 2.5 mM phosphoenol pyruvate, 0.5 units pyruvate kinase and 39–157 nM wild-type E. coli EF-Tu or 33–130 nM E. coli EF-Tu E287R. Following a 10 min incubation at 37˚C, 5 mM ATP was added to each reaction to displace the [γ-32P]GTP bound to pyruvate kinase to decrease the background. The reactions were analyzed using a filter binding assay.24 The binding of GTP was corrected for the retention of radiolabeled GTP in a minus EF-Tu control (approximately 0.3 pmol).

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Figure 2. Activity of EF-Tumt R335E in ternary complex. (A) Ternary complex formation was assayed by examining the ability of 0.1–0.2 µM EF-Tumt (o) and EF-Tumt R335E (O) to protect 0.1 µM mitochondrial [14C]Phe-tRNAPhe from hydrolysis by RNase A as described in ‘Materials and Methods’. All concentrations are corrected for the percentage of EF-Tu molecules active in ternary complex formation with E. coli Phe-tRNAPhe. (B) Ternary complex formation with 0.31 µM E. coli [14C]Phe-tRNAPhe and 0.05–0.28 µM wild-type EF-Tumt (o) or EF-Tumt R335E (O).

RESULTS Rationale. Domain II of EF-Tu is essential for the stable binding of the aa-tRNA to the factor during ternary complex formation. Most residues involved in aa-tRNA binding are highly conserved in EF-Tu (Fig 1). However, there are a few residues that lack conservation among the prokaryotic and mammalian mitochondrial factors that may be important in ternary complex formation. One such residue, Glu287 (unless otherwise indicated, numbering is that of E. coli EF-Tu), is adjacent to the positivelycharged binding pocket for the 5’ end of the aa-tRNA. Glu287 (Arg335 in EF-Tumt) also borders the highly conserved Arg288 that forms a salt bridge to the 5’ phosphate of the aa-tRNA. The loop containing Glu287 is involved in stabilizing the binding of the 5’ end of the aa-tRNA to EF-Tu.2,3 Alignments of mitochondrial and prokaryotic factors indicate that the residue at position 287 is negatively-charged (or is the amide of a negatively charged residue) in prokaryotic EF-Tu but is a positively charged residue in the equivalent position in animal mitochondrial factors (Fig. 1). The difference in the characteristics of residue 287 in prokaryotic and mitochondrial EF-Tu may influence the properties of each factor, including its interaction with prokaryotic and mitochondrial aa-tRNAs. Activity of EF-Tumt R335E in Polymerization. Wild-type EF-Tumt and its R335E variant were expressed in E. coli as His-tagged proteins and purified by affinity chromatography to near homogeneity (data not shown). The variant was expressed at about 90% of the level of the wild-type factor. Evaluation of the properties of mutated forms of EF-Tumt requires an assessment of the percentage of active molecules in the preparation.19,20,29 For EF-Tumt this percentage of active molecules is generally measured by determining the fraction of the factor that can form a ternary complex with aa-tRNA.23 In general, about 40% of the EF-Tumt forms ternary complexes.23 The R335E variant of EF-Tumt was as active as the wild-type factor in this assay indicating that the mutation was not affecting the basic fold of the protein. The ability of EF-Tumt R335E to function in poly(U)-directed polymerization of [14C]Phe was compared to that of the wild-type factor using E. coli Phe-tRNAPhe and E. coli ribosomes and in assays using mitochondrial Phe-tRNAPhe and mitochondrial ribosomes. In both of these assays, the R335E variant displayed an activity similar to that observed with the wild-type factor (data not shown). Thus, this mutation does not affect the ability of the variant to function in in vitro translation. However, activity in the polymerization assay basically reflects the rate limiting step in the overall process which is thought to be the release of EF-Tu•GDP from the ribosome.30 Hence, the properties of the R335E variant of EF-Tumt were examined in more specific assays. 98

Ternary Complex Formation with EF-Tumt R335E. Noncanonical mitochondrial aa-tRNAs are less stable and may form different interactions with EF-Tu than their cytoplasmic and prokaryotic counterparts.8 Previous work31 has shown that the ternary complex formed with EF-Tumt and mitochondrial Phe-tRNAPhe is less stable (KD, 76 nM) than that with E. coli Phe-tRNAPhe (KD, 18 nM). The ability of EF-Tumt R335E to form a ternary complex with mitochondrial Phe-tRNAPhe was evaluated as described in ‘Materials and Methods’ and compared to that of the wild-type factor. As indicated in Figure 2A, EF-Tumt R335E shows a drastic reduction in its ability to form a ternary complex with mitochondrial aa-tRNA when compared to wild-type EF-Tumt. Analysis of this data suggests that the KD for the ternary complex formed between EF-Tumt R335E and mitochondrial Phe-tRNAPhe is about 10-fold higher (about 600 nM) than that observed with the wild-type factor. Hence, Arg335 is apparently important in stabilizing the interactions between EF-Tumt and mitochondrial Phe-tRNAPhe. This observation is compatible with the placement of the conserved the Arg residue in the pocket that binds the 5’ end of the aa-tRNA in the ternary complex. The interaction between EF-Tu and aa-tRNA can be monitored by a nonenzymatic hydrolysis protection assay as well as the RNase protection assay used above.32 The nonenzymatic hydrolysis protection assay measures the accessibility of the ester bond between the aa-tRNA while the RNase protection assay monitors the sensitivity of the polynucleotide backbone of the aa-tRNA. There are situations in which these two assays provide different pictures of the interaction of EF-Tu and aa-tRNA.32 The nonenzymatic hydrolysis protection assay was used to examine the interaction of wild type EF-Tumt and EF-Tumt R335E with mitochondrial Phe-tRNAPhe. In these experiments the half life (t1/2) of the complex between EF-Tumt and mitochondrial Phe-tRNAPhe is 330 min while the t1/2 between EF-Tumt R335E and Phe-tRNAPhe is only 100 min. Thus, both assays indicate that EF-Tumt R335E is deficient in interacting with mitochondrial Phe-tRNAPhe. To determine whether the results observed with mitochondrial Phe-tRNAPhe were specific for mitochondrial aa-tRNAs, ternary complex formation was examined using E. coli Phe-tRNAPhe. Interestingly, in the RNase protection assay, EF-Tumt R335E is as active as wild-type EF-Tumt in ternary complex formation assays with E. coli Phe-tRNAPhe (Fig. 2B). However, in the nonenzymatic hydrolysis protection assay, the complex between EF-Tumt R335E and E. coli Phe-tRNAPhe appears to be somewhat less stable than that observed with wild-type EF-Tumt with a t1/2 of 550 min compared to 1200 min for the wild-type factor. Thus, Arg335 appears to have a subtle effect on the complex formed between EF-Tumt R335E and E. coli Phe-tRNAPhe. One possible explanation is that this mutation leads to a small change in the binding pocket for the amino acid making this region more accessible to -OH and, therefore, leading to a somewhat greater hydrolysis of the ester bond.

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a filter binding assay whereas that of EF-Tumt cannot. Guanine nucleotide binding to EF-Tu occurs on Domain I and the E287R mutation created here is located in Domain II. However, there are contacts between Domain I and Domain II especially in the GTP-bound form of the factor. In addition, Domains II and III affect the binding constant for guanine nucleotide binding to EF-Tu.36 The ability of E. coli EF-Tu E287R to interact with GDP and GTP was, therefore, measured and was compared to the wild-type prokaryotic factor. EF-Tu was purified in the presence of GDP. The excess GDP was separated from EF-Tu by chromatography on a Sephadex G-50 column (data not shown) and the concentration of active EF-Tu in each sample was determined immediately Figure 3. Activity of EF-Tumt R335E in binding Phe-tRNAPhe to the A-site of the ribosome. To determine after chromatography in a filter binding assay. The the ability of wild-type and variant EF-Tu to deliver Phe-tRNAPhe to the A-site of the ribosome, assays abilities of wild-type and variant E. coli EF-Tu to were carried out using the nonhydrolyzable analogue of GTP, GDPNP, as described in ‘Materials and bind GDP were measured with a limiting amount Methods’. (A) 45–90 nM wild-type EF-Tumt (o) or EF-Tumt R335E (O) with mitochondrial Phe-tRNAPhe. of [3H]GDP. The affinity of E. coli EF-Tu E287R (B) 23–115 nM wild-type EF-Tumt (o) or 22 to 110 nM EF-Tumt R335E (O) with E. coli [14C]Phe-tRNAPhe. for GDP (KD, 11 nM) is similar to that of the wild-type factor (KD, 10 nM) in such GDP binding assays (Fig. 4A). Both values are comparable to A-site Binding Activity of EF-Tumt R335E. The results presented above that of a previous study (KD, 7.7 nM).25 The dissociation rate constant of the EF-Tu•GDP complex was also clearly indicate that EF-Tumt R335E interacts with mitochondrial Phe-tRNAPhe less effectively than does the wild-type factor. To examine the determined for the wild-type and variant E. coli factors as described in ability of this variant to deliver this aa-tRNA to the ribosome, its activity in ‘Materials and Methods’. The prokaryotic variant releases GDP at a rate A-site binding assays was examined directly. These assays were carried out in similar to that of the wild-type factor (Fig. 4B). In addition, the koff values the presence of excess GDPNP, a nonhydrolyzable analogue of GTP allowing of wild-type E. coli EF-Tu (5.0 x 10-3/s ) and E. coli EF-Tu E287R (5.1 x 10-3/s) measured at 37˚C resemble those previously determined at 30˚C only a single round of ribosome binding per active EF-Tu molecule. In assays using mitochondrial Phe-tRNAPhe and E. coli ribosomes, (4.4 x 10-3/s).27 In the crystal structure of the EF-Tu•GDPNP complex, Arg288 is EF-Tumt R335E shows a substantial decrease in A-site binding activity compared to wild-type EF-Tumt (Fig. 3A). This decrease is over and above involved in the formation of a network of H-bonds connecting the switch that observed in ternary complex formation. It is important to note that the regions of Domain I to the rest of the molecule.37,38 Arg288 is in direct amount of ternary complex formed with mitochondrial Phe-tRNAPhe contact with the switch II region of Domain I through a H-bond to Asn90, exceeds that of the level of A-site binding (Fig. 2A) for the variant and which in turn H-bonds to Ile63 in the switch I region. The interaction of wild-type factors. Thus, it is apparent that ternary complex formation is not the aa-tRNA with EF-Tu disrupts the H-bond between Arg288 and the limiting in the assay. In addition, A-site binding assays were carried out switch II region and Arg288 is repositioned to form a salt-bridge to the using preformed ternary complexes and similar results were obtained (data 5’ phosphate of the aa-tRNA. It is possible that the E287R mutation intronot shown). Thus, Arg335 is important both for ternary complex formation duced into E. coli EF-Tu alters the interactions of the neighboring Arg288 with the switch regions of Domain I possibly affecting the binding of GTP and for the binding of the ternary complex to the ribosome. The activities of wild-type EF-Tumt and the R335E variant in A-site with the E287R variant. The ability of the E287R variant of E. coli EF-Tu to form a binary binding using E. coli Phe-tRNAPhe and E. coli ribosomes were measured and compared to those of the respective wild-type factor (Fig. 3B). With E. coli complex with [γ-32P]GTP was determined using a filter binding assay and Phe-tRNAPhe, the R335E variant is as active as the wild-type factor in compared to that of the wild-type factor. As indicated in Figure 4C, the promoting the binding of the aa-tRNA to the A-site. This observation is in prokaryotic variant is similar to wild-type E. coli EF-Tu in GTP binding agreement with the ability of the variant to form a ternary complex with the activity. Thus, the E287R mutation introduced into E. coli EF-Tu does not bacterial aa-tRNA as effectively as the wild-type factor. Thus, the Arg at affect the ability of the factor to interact with guanine nucleotides. position 335 is not important for the binding of the ternary complex Ternary Complex Formation of E. coli E287R: The observation reported formed with E. coli Phe-tRNAPhe to the A-site. above indicated that mutation of Arg335 in EF-Tumt reduces the ability of Activity of E. coli E287R in Polymerization. The wild-type and E287R this factor to form a ternary complex with mitochondrial Phe-tRNAPhe. variant of E. coli EF-Tu were expressed as His-tagged proteins and purified Hence, it was of interest to determine whether the mutation of the correto near homogeneity (data not shown). The variant was expressed essentially sponding Glu287 in E. coli EF-Tu to Arg would improve its interaction with as well as the wild-type factor indicating that it did not have any funda- mitochondrial aa-tRNA. Interestingly, using the RNase protection assay, the mental problems with folding as a result of the mutation. The percentage of E287R variant shows a measurable improvement in its ability to form a E. coli EF-Tu E287R active in GDP binding assays was compared to that of ternary complex with mitochondrial Phe-tRNAPhe (Fig. 5A). This observawild-type E. coli EF-Tu and was essentially identical to that of the wild-type tion is in agreement with the apparent decrease in ternary complex formafactor (approximately 80%). In addition, this variant has approximately the tion when the corresponding Arg335 was mutated in EF-Tumt (Fig. 2A). same percentage of molecules active in ternary complex formation as the The nonenzymatic hydrolysis protection assay supports the observation respective wild-type factor (about 30%, data not shown). This value is made with the RNase protection assay. The t1/2 for the E. coli EF-Tu E287R variant complexed with mitochondrial Phe-tRNAPhe is 770 min compared consistent with observations from other laboratories.25,33 Interactions of E. coli EF-Tu E287R with Guanine Nucleotides. E. coli to a t1/2 of just over 200 min for the wild-type factor. EF-Tu has a higher affinity for GDP (KD 7.7 nM) and GTP (KD 0.3 µM) The ability of the E287R variant to bind E. coli Phe-tRNAPhe was tested than EF-Tumt (KD 1.0 µM and 18 µM respectively).25,34,35 Hence, the to determine whether it might also show an increased activity in ternary affinity of E. coli EF-Tu for either guanine nucleotide can be measured using complex formation. As indicated in Figure 5B, in the RNase protection

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A

A

B

B

C

C

Figure 4. Interaction of E. coli EF-Tu E287R with guanine nucleotides. (A) The affinity of E. coli EF-Tu E287R (l) towards GDP was determined in a filter binding assay using limiting amounts of [3H]GDP and compared to that of the wild-type factor (n). (B) The dissociation of GDP from the E. coli EF-Tu• [3H]GDP complex was monitored over time for both wild-type (n) and variant (l) factors and plotted as ln(xt/x0) as a function of time as described in ‘Materials and Methods’. (C) The ability of wild-type E. coli EF-Tu (n) and E. coli EF-Tu E287R (l) to bind [γ-32P]GTP was monitored as described in ‘Materials and Methods’.

assay, the variant binds the prokaryotic Phe-tRNAPhe with the same affinity as does the wild-type factor. Similar results were observed with EF-Tumt R335E and E. coli EF-Tu E287R using E. coli Cys-tRNACys (data not shown). In the nonenzymatic hydrolysis protection assay, both variant and wild-type bacterial factors show the same t1/2 of about 1100 min under the assay conditions used. Therefore, to further assess the interaction between the wild-type and variant E. coli EF-Tu, the rate at which E. coli Phe-tRNAPhe dissociates from the EF-Tu•GTP complex was monitored (Fig. 5C). Surprisingly, the complex involving E. coli EF-Tu E287R had a significantly slower dissociation rate constant (koff, 4.8 x 10-4/s) than that

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Figure 5. Activity of E. coli E287R in Ternary Complex Formation: Ternary complex formation was assayed by examining the ability of EF-Tu to protect Phe-tRNAPhe from hydrolysis by RNase A as described in ‘Materials and Methods’. (A) 0.09–0.17 µM wild-type E. coli EF-Tu (n) and 0.09–0.20 µM E. coli EF-Tu E287R (l) were tested for the ability to form ternary complexes with 0.1 µM mitochondrial Phe-tRNAPhe following a 2–2.5 h incubation at 37˚C in the presence of phosphoenol pyruvate and pyruvate kinase to remove the GDP contained in the EF-Tu preparation. (B) Wild-type E. coli EF-Tu and E. coli EF-Tu E287R prepared in 50 µM GTP were incubated 2.5 h at 37˚C in a buffer containing phosphoenol pyruvate and pyruvate kinase. Following this incubation, the activities of the factors were evaluated by examining the abilities of 0.02–0.08 µM wild-type E. coli EF-Tu (n) and 0.03–0.09 µM E. coli EF-Tu E287R (l) to protect 0.08 µM E. coli Phe-tRNAPhe from hydrolysis. (C) The dissociation of [14C]Phe-tRNAPhe from wild-type E. coli EF-Tu (n) and E. coli EF-Tu E287R (l) complexed with GTP was monitored over time following a 2.5 h incubation at 37˚C in the presence of phosphoenol pyruvate and pyruvate kinase as indicated in ‘Materials and Methods’.

observed for wild-type E. coli EF-Tu (koff, 1.7 x 10-3/s). This latter value is comparable to that obtained in a previous study.25 Since there is no apparent change in the binding constant for the ternary complex as a result of the

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A-site binding assays were carried out directly. The ability of the E287R variant to deliver mitochondrial [14C]Phe-tRNAPhe to the A-site of ribosomes was determined as described in ‘Materials and Methods’ and compared to that of the wild-type factor. As indicated in Figure 6A, the activity of E. coli EF-Tu E287R in this assay is significantly greater than that of the wild-type prokaryotic factor (Fig. 6A). Apparently, Arg335 enhances the ability of EF-Tumt to deliver mitochondrial aa-tRNA to the ribosome while the presence of Glu287 in E. coli EF-Tu reduces the activity of the factor in such A-site binding assays. It is important to note here that the amount of ternary complex formed by both the wild-type and E287R variant of E. coli EF-Tu is greater than the amount of A-site binding achieved (compare Figure 6. Activity of E. coli EF-Tu E287R in binding Phe-tRNAPhe to the A-site of the ribosome. A-site Fig. 5A to Fig. 6A). Hence, the amount of A-site binding assays were performed as described in ‘Materials and Methods’ to determine the ability of binding achieved by the E. coli factor is not limitwild-type and variant EF-Tu to deliver Phe-tRNAPhe to the ribosome. The pmol of EF-Tu indicated are ed by the availability of the ternary complex. corrected for the percentage of EF-Tu molecules active in ternary complex formation. (A) Activity of The activity of the E287R variant was also 50–100 nM wild-type E. coli EF-Tu (n) and E. coli EF-Tu E287R (l) in binding mitochondrial tested in A-site binding assays with E. coli [14C]Phe-tRNAPhe to the A-site of the ribosome. (B) Activity of 14–80 nM wild-type E. coli EF-Tu (n) or Phe-tRNAPhe (Fig. 6B). As expected, this variant E. coli EF-Tu E287R (l) in binding E. coli [14C]Phe-tRNAPhe to the A-site of the ribosome. was indistinguishable from the wild-type factor under these conditions. This data indicates that the Glu at position 287 of E. coli EF-Tu does not play an essential role in the EF-Tu-mediated delivery of E. coli Phe-tRNAPhe to the A-site of the ribosome.

DISCUSSION

Figuer 7. Role of residue 287 of Domain II on the interaction of EF-Tu with aa-tRNA. The Insight II Molecular Modeling Package program (http:// www.accelrys.com) was used to display the interaction of residue 287 (E. coli numbering) of Domain II with Cys-tRNACys. Residue 287 is purple; residue 288 is blue; the remainder of Domain II is yellow; G1 of the tRNA is red; the remainder of the tRNA is green. The coordinates were obtained from the crystal structure of the ternary complex T. aquaticus EF-Tu•GDPNP•E. coli Cys-tRNACys, PDB# 1B23. Gln287 corresponds to Gln299 in T. aquaticus EF-Tu.

mutation, the kon value for ternary complex formation is most likely also decreased in the E287R variant. This observation suggests that the presence of the Glu residue at position 287 increases the forward rate constant for ternary complex formation. However, once the aa-tRNA is bound in the ternary complex, the Glu also permits its more rapid release. In contrast, an Arg residue at this position apparently reduces both the on rate and the off rate giving rise to the same equilibrium dissociation constant. A-site Binding Activity of E. coli. E287R. Previous studies have shown that E. coli EF-Tu is not as active as EF-Tumt in binding mitochondrial Phe-tRNAPhe to the A-site of the ribosomes.10 In an effort to determine if Arg335 contributes to the higher activity of EF-Tumt observed in this assay,

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Glu287 is adjacent to the highly conserved residue Arg288 that forms a salt bridge to the oxygen4 atom of the phosphate group of G1 of the aa-tRNA (Fig. 7). Arg288 is an important residue that stabilizes the binding of the 5’ end of the aa-tRNA to EF-Tu.22 Mutation of Arg288 reduces the formation of the ternary complex significantly. Removal of the 5’ phosphate on the aa-tRNA allows ternary complex formation but reduces the efficiency of the aa-tRNA in polymerization.39 The residues that comprise the binding pocket for the 5’ end of the aa-tRNA are highly conserved and have a defined charge distribution. The purpose of this charge distribution is presumably to direct the negatively- charged phosphate group at the 5’ end of the incoming aa-tRNA into its binding site.2,3 Unlike in E. coli EF-Tu, the position equivalent to Glu287 in the binding pocket for the 5’ end of the aa-tRNA is held by a basic residue in EF-Tumt. This residue lies just outside of what is designated in bacterial EF-Tu as the positively-charged binding pocket for the 5’ end of the aa-tRNA. The Arg335 residue of EF-Tumt alters the charge balance of the amino acids outlining this pocket and perhaps serves as an additional stabilizing residue in the binding pocket of the mitochondrial factor. This additional stabilization would presumably enhance the binding of the more conformationally fragile mitochondrial aa-tRNAs to the factor. The ternary complexes formed between EF-Tu and mitochondrial aa-tRNA are generally weaker than those formed with canonical aa-tRNAs.31 In the ternary complex, EF-Tu interacts with the -CCA end carrying the amino acid, the acceptor stem including the 5’ phosphate and with the T-stem of the aa-tRNA. Mitochondrial tRNAs often have unpaired residues in their stem regions that could impair the interactions between the aa-tRNA and EF-Tu. These mismatches certainly affect the ordered structure of the A-helix of the aa-tRNA that contacts EF-Tu. For example, bovine mitochondrial Phe-tRNAPhe has a G:A mismatch in the acceptor stem and only 4 base pairs in the T-stem.40

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Presumably, the unusual structure of this tRNA reduces its affinity for EF-Tumt (KD, 76 nM) compared to that of the canonical E. coli Phe-tRNAPhe (KD, 18 nM).23,31 Ternary complex formation assays indicate that replacing Arg335 in EF-Tumt with an acidic residue disrupts the binding of the factor to mitochondrial Phe-tRNAPhe (Fig. 2A). Thus, it is reasonable to suppose that Arg335 might actually make a second electrostatic contact with the 5’ phosphate of mitochondrial Phe-tRNAPhe similar to that observed between Arg288 of E. coli EF-Tu and bacterial aa-tRNA (Fig. 7). This interaction is lost in the R335E variant resulting in a significant decrease in stability of the ternary complex. Interestingly, replacing Glu287 in E. coli EF-Tu with Arg enhances the activity of the prokaryotic factor in ternary complex formation with mitochondrial Phe-tRNAPhe. This result supports the idea that placement of the basic residue at the equivalent position in E. coli EF-Tu serves to enhance the stability of the ternary complex formed with mitochondrial aa-tRNA. Furthermore, the E287R mutation increases the ability of E. coli EF-Tu to deliver mitochondrial Phe-tRNAPhe to the A-site of the ribosome. In fact, this increase in the activity of the prokaryotic factor brings the activity of E. coli EF-Tu with mitochondrial Phe-tRNAPhe closer to that observed with EF-Tumt. Thus, it is clear that the residue at position 335 in EF-Tumt is important for ternary complex formation and A-site binding with mitochondrial Phe-tRNAPhe. References 1. Krab I, Parmeggiani A. EF-Tu, a GTPase odyssey. Biochim Biophys Acta 1998; 1443:1-22. 2. Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L, Clark B, et al. Crystal structure of the ternary complex of Phe-tRNAphe, EF-Tu and a GTP analog. Science 1995; 270:1464-72. 3. Nissen P, Thirup S, Kjeldgaard M, Nyborg J. The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Structure 1999; 7:143-56. 4. Song H, Parsons MR, Rowsell S, Leonard G, Phillips SE. Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 Å resolution. J Mol Biol 1999; 285:1245-56. 5. Kawashima T, Berthet-Colominas C, Wulff M, Cusack S, Leberman R. The structure of the Escherichia coli EF-Tu:EF-Ts complex at 2.5 Å resolution. Nature 1996; 379:511-8. 6. Andersen G, Thirup S, Spremulli LL, Nyborg J. High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP. J Mol Biol 2000; 297:421-36. 7. Helm M, Brule H, Friede D, Giege R, Putz D, Florentz C. Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA 2000; 6:1356-79. 8. Grosjean H, Cedergren RJ, McKay W. Structure in tRNA data. Biochimie 1982; 64:387-97. 9. Hayashi I, Kawai G, Watanabe K. Higher-order structure and thermal instability of bovine mitochondrial tRNASerUGA investigated by proton NMR spectroscopy. J Mol Biol 1998; 284:57-69. 10. Kumazawa Y, Schwartzbach C, Liao H-X, Mizumoto K, Kaziro Y, Watanabe K, et al. Interactions of bovine mitochondrial phenylalanyl-tRNA with ribosomes and elongation factors from mitochondria and bacteria. Biochim Biophys Acta 1991; 1090:167-72. 11. Schwartzbach C, Spremulli LL. Interaction of animal mitochondrial EF-Tu:EF-Ts with aminoacyl-tRNA, guanine nucleotides and ribosomes. J Biol Chem 1991; 266:16324-30. 12. Bullard JM, Cai Y-C, Zhang Y, Spremulli LL. Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes. Biochim Biophys Acta 1999; 1446:102-14. 13. Schwartzbach C, Spremulli LL. Bovine mitochondrial protein synthesis elongation factors: Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex. J Biol Chem 1989; 264:19125-31. 14. Eberly SL, Locklear V, Spremulli LL. Bovine mitochondrial ribosomes. Elongation factor specificity. J Biol Chem 1985; 260:8721-5. 15. Woriax V, Bullard JM, Ma L, Yokogawa T, Spremulli LL. Mechanistic studies of the translational elongation cycle in mammalian mitochondria. Biochim Biophys Acta 1997; 1352:91-101. 16. Zhang Y, Li X, Spremulli LL. Role of the conserved aspartate and phenylalanine residues in prokaryotic and mitochondrial elongation factor Ts in guanine nucleotide exchange. FEBS Lett 1996; 391:330-2. 17. Bullard JM, Cai Y-C, Spremulli LL. Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase. J Mol Biol 1999; 288:567-77. 18. Woriax V, Burkhart W, Spremulli LL. Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu. Biochim Biophys Acta 1995; 1264:347-56.

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19. Hunter SE, Spremulli LL. Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu. Biochim Biophys Acta 2004; 1699:173-82. 20. Hunter SE, Spremulli LL. Mutagenesis of Gln290 of Escherichia coli and mitochondrial elongation factor Tu affects interactions with mitochondrial aminoacyl-tRNA and GTPase activity. Biochem 2004; 43:6917-27. 21. Pingoud A, Urbanke C. The determination of binding parameters from protection experiments. Anal Biochem 1979; 92:123-7. 22. Rattenborg T, Nautrup-Pedersen G, Clark BFC, Knudsen C. Contribution of Agr288 of Escherichia coli elongation factor Tu to translational functionality. Eur J Biochem 1997; 249:408-14. 23. Cai Y-C, Bullard JM, Thompson NL, Spremulli LL. Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts. J Biol Chem 2000; 275:20308-14. 24. Ravel JM, Shorey RL, Froehner S, Shive W. A study of the enzymic transfer of aminoacyl-RNA to Escherichia coli ribosomes. Arch Biochem Biophys 1968; 125:514-26. 25. Louie A, Jurnak F. Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5’-triphosphate-aminoacyl-tRNA complexes. Biochem 1985; 24:6433-9. 26. Parmeggiani A, Sander G. Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2. Mol Cell Biochem 1981; 35:129-58. 27. Fasano O, Crechet J-B, Parmeggiani A. Preparation of nucleotide-free elongation factor Tu and its stabilization by the antobiotic kirromycin. Anal Biochem 1982; 124:53-8. 28. Knudsen C, Clark BFC. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: Effects on the GTPase reaction and aminoacyl-tRNA binding. Protein Engin 1995; 8:1267-73. 29. Bilgin N, Kirsebom LA, Ehrenberg M, Kurland CG. Mutations in ribosomal proteins L7/L12 perturb EF-G and EF-Tu functions. Biochimie 1988; 70:611-8. 30. Pape T, Wintermeyer W, Rodnina MV. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome. EMBO J 1998; 17:7490-7. 31. Hunter SE, Spremulli LL. Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNAs. Mitochondrion 2004; In press. 32. Jonak J, Karas K. Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L-phenylalanyl chloromethane abolishes its ability to interact with the 3’-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA. FEBS Lett 1989; 251:121-4. 33. Pleiss JA, Uhlenbeck OC. Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA. J Mol Biol 2001; 308:895-905. 34. Miller D, Weissbach H. Studies on the purification and properties of factor Tu from E. coli. Arch Biochem Biophys 1970; 141:26-37. 35. Cai Y-C, Bullard JM, Thompson NL, Spremulli LL. Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides. Prot Sci 2000; 9:1791-800. 36. Jensen M, Cool R, Mortensen K, Clark B, Parmeggiani A. Structure-function relationships of elongation factor Tu: Isolation and activity of the guanine-nucleotide-binding domain. Eur J Biochem 1989; 182:247-55. 37. Berchtold H, Reshetnikova L, Reiser C, Schirmer N, Sprinzl M, Hilgenfeld R. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 1993; 365:126-32. 38. Kjeldgaard M, Nissen P, Thirup S, Nyborg J. The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure 1993; 1:35-50. 39. Sprinzl M, Graeser E. Role of the 5’-terminal phosphate of tRNA for its function during protein biosynthesis elongation cycle. Nuc Acids Res 1980; 8:4737-44. 40. Sprinzl M, Hartmann T, Weber J, Blank J, Zeidler R. Compilation of tRNA sequences and sequences of tRNA genes. Nuc Acids Res 1989; 17:1-172.

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