Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP

A new approach for the fluorescence labeling of an aminoacyl-tRNA at the 3'-end is applied to study its interaction with bacterial elongation factor Tu (EF-Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr-C-C-A was replaced by 2-thiocytidine (s2C). The resulting tRNATyr-C-s2C-A was aminoacylated and then alkylated at the s2C residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS). A greater than 100% increase in the intensity of fluorescence emission of the modified Tyr-tRNATyr-C-s2C(AEDANS)-A was observed upon interaction with EF-Tu.GTP. A ternary complex dissociation constant of 1.27 X 10(-8) M was calculated from this direct interaction. Using such fluorescent aminoacyl-tRNA, the affinity of any unmodified aminoacyl-tRNA can be determined by competition experiments. By this approach, we show here that the affinity of unmodified Tyr-tRNATyr-C-C-A is identical to that of the modified Tyr-tRNATyr. This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl-tRNA for EF-Tu.GTP. The introduction of 2-thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid-protein interactions.     

The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.     

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins. Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA.     

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.     

Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA.

Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.     

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.     

The site of interaction of aminoacyl-tRNA with elongation factor Tu

We have used RNases T1, T2 and A to digest two aminoacyl-tRNAs, Escherichia coli Phe-tRNAPhe and E. coli Met- tRNAMetm both in the naked forms and in ternary complexes with E. coli elongation factor Tu (EF-Tu) and GTP. An analysis of the 'footprinting' results has led to an interpretation that has localized the part of the three-dimensional structure of aminoacyl-tRNA covered by the protein in the ternary complex. In terms of the three-dimensional structure of tRNA established for yeast tRNAPhe, EF-Tu covers the aa-end, aa-stem, T-stem, and extra loop on the side of the L-shaped tRNA that exposes the extra loop.     

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu

The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.     

Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP

gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.     

Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA

The ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.     

Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis.

The reaction of fluorescamine with primary amino groups of tRNAs was investigated. The reagent was attached under mild conditions to the 3'-end of tRNAPhe-C-C-A(3'NH) from yeast and to the minor nucleoside x in E. coli tRNAArg, tRNALys, tRNAMet, tRNAIle and tRNAPhe. The primary aliphatic amino groups of these tRNAs react specifically so that the fluorescamine dye is not attached to the amino groups of the nucleobases. E. coli tRNA species modified on the minor nucleoside X47 can all be aminoacylated. An involvement of the minor modified nucleoside X47 in the tRNA: synthetase interaction is detected. Native tRNALys-C-C-A from E. coli can be phenylalanylated by phenylalanyl-tRNA synthetase from yeast, whereas this is not the case for fluorescamine treated tRNALys-C-C-A(XF47). Pre-tRNAPhe-C-C-A(XF47) forms a ternary complex with the elongation factor Tu:GTP from E. coli, binds enzymatically to the ribosomal A-site and is active in poly U dependent poly Phe synthesis. Fluorescamine-labelled E. coli tRNAs provide new substrates for the study of protein biosynthesis by spectroscopic methods.     

Ternary complex between elongation factor Tu•GTP and Phe-tRNA

The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNA^Phe was examined by ¹H-NMR spectroscopy. Esterification of phenylalanine to tRNA^Phe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA^Phe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA^Phe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA^Phe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA^Phe preserves its typical L-shaped tertiary structure in the complex. At high (> 10⁻⁴ M) ligand concentrations a complex between tRNA^Phe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA^Phe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.     

The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ.

The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated. As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA. Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli. The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis. Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64. Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.     

Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies

The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Elongation factor Tu can reduce translation errors in poly(U)-directed cell-free systems

Rates of incorporation of [³H]phenylalanine and [¹⁴C]leucine from the aminoacylated transfer-RNA into polypeptides synthesized on poly(U) programmed Escherichia coli ribosomes have been determined in cell-free translation systems containing either elongation factors Tu and G with GTP, or just elongation factor Tu or G with GTP, or none of the elongation factors. The presence of elongation factor Tu with GTP has been shown to reduce the leucine to phenylalanine ratio in the product at relatively low concentrations of Mg²⁺. This error-reducing effect of elongation factor Tu has not been observed at high concentrations of Mg²⁺, although the factor still contributed to the speed of elongation. The results are discussed in terms of the kinetic proof-reading mechanism proposed by Hopfield (1974).     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu

The products of nitrous acid mediated-deamination of Phe-tRNAPhe from E. coli were analyzed and their capability to interact with elongation factor Tu from E. coli was investigated. Thin-layer chromatography as well as HPLC analysis revealed the existence of at least two deamination products, 3-phenyl-lactyl-tRNAPhe and cinnamyl-tRNAPhe. It could be shown that the aminoacyl-tRNA analogues were active in the formation of the ternary complex with EF-Tu X GTP, although with a lower efficiency than native Phe-tRNAPhe. For both modified acyl-tRNAs the dissociation constant was determined to be 3 X 10(-5) M.     

Affinities of tRNA binding sites of ribosomes from Escherichia coli

The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.