The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis

The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA. By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA. These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA. We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions. These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation. The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome. These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.     

Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.     

Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes

The mammalian mitochondrial complex consisting of elongation factors EF-Tu and EF-Ts (EF-Tu.Tsmt) is capable of efficiently binding aminoacyl-tRNA to the ribosome in the presence and absence of guanine nucleotides. In the presence of GTP the binding reaction is catalytic. In the absence of guanine nucleotides, or in the presence of a non-hydrolyzable GTP analog, only one round of ribosome binding occurs. EF-Tu.Tsmt is capable of forming a ternary complex with GTP and Escherichia coli Phe-tRNA as demonstrated by gel filtration chromatography, nitrocellulose filter binding, and by protection of the aminoacyl-tRNA bond from hydrolysis. GDP and the non-hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate are also capable of facilitating ternary complex formation with EF-Tu.Tsmt, but are less effective. No kinetic advantage results from the formation of this ternary complex prior to ribosome binding, and EF-Tu.Tsmt may actually bind aminoacyl-tRNA directly to the ribosome prior to binding GTP. These results suggest that a variation of the prokaryotic elongation cycle is occurring in animal mitochondria. N-Ethylmaleimide inhibits the activity of EF-Tu.Tsmt in polymerization and in ribosome binding. However, the activity of the EF-Tsmt which can be measured independently, is not altered.     

Guanosine 5'-O-(3-thiotriphosphate) as an analog of GTP in protein biosynthesis. The effects of temperature and polycations on the accuracy of initial recognition of aminoacyl-tRNA ternary complexes by ribosomes

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) is a good analog of GTP in the reactions leading to the formation of a peptide bond in protein biosynthesis. It forms binary and ternary complexes with elongation factor Tu (EF-Tu), and with EF-Tu and aminoacyl-tRNA (aa-tRNA). In addition, it stimulates aa-tRNA binding to ribosomes. Although GTP gamma S hydrolysis is more than three orders of magnitude slower than GTP hydrolysis, both reactions are dependent on the formation of a noncovalent complex (RS X TC) between mRNA-programmed ribosomes and ternary complex, and the complexes resulting from that hydrolysis are intermediates in peptide formation. The rate of dissociation of the ribosome X EF-Tu X GTP gamma S X aa-tRNA complex was determined from the rate of labeled peptide formation in the presence of an unlabeled ternary complex chase. This rate (2.2 X 10(-3) s-1) is similar to that determined previously (Thompson, R.C., and Karim, A.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4922-4926) from the progress of GTP gamma S hydrolysis. The effects of temperature and polycation concentration on this rate constant and that for GTP gamma S hydrolysis are reported. The rate constants measured are consistent with a kinetic rather than thermodynamic limit on the accuracy of the aa-tRNA selection in vivo.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ppGpp on the accuracy of protein biosynthesis

The maintenance of accuracy in protein biosynthesis in amino acid-starved rel+ strains of Escherichia coli has been attributed to an effect of ppGpp on the accuracy of aa-tRNA selection by the ribosome. It has been determined that concentrations of ppGpp characteristic of those found in amino acid-starved cells have no effect on the rate of reaction of poly(U)-programmed ribosomes with either the cognate (Phe) or the near-cognate (Leu2) ternary complexes. Neither the rate of GTP hydrolysis, which signals selection of the ternary complex, nor the rate of peptide formation, which signals the acceptance of the aa-tRNA after proofreading, is affected by the nucleotide. The results indicate that the effect of ppGpp in maintaining the accuracy of protein biosynthesis in cells starved for an amino acid is not due to a direct effect on the rate constants for substrate selection by the ribosome.     

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 . GTP . aminoacyl-tRNA, is necessary for the formation of a ribosome . EF-1 complex. However, when gel filtration was used to isolate the ribosome . EF-1 complex, only template and tRNA were required. These studie emphasize the fact that the procedure used to isolate the ribosome . EF-1 complex determines the requirements for stable complex formation. EF-1 can also interact with nucleic acids such as 28 S and 18 S rRNA, messenger RNA and DNA. In contrast to the binding to ribosomes, EF-1 binding to nucleic acids requires only Mg2+.     

tRNA selection and kinetic proofreading in translation

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.     

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.     

Kinetic determinants of high-fidelity tRNA discrimination on the ribosome

The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading. Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally. Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex. tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate.     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond 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.     

On the mechanism of a calcium-associated defect of oxidative phosphorylation in progessive muscular dystrophy

Partially purified elongation factor 1 preparations from calf brain, sheep brain, calf liver, and rabbit reticulocytes have been compared in their ability to interact with GTP and Phe-tRNA. A nitrocellulose filter assay has been used to study these interactions, and with all the EF1 preparations studied, evidence has been obtained for the formation of a Phe-tRNA·-EF1·-GTP complex. The ternary complex reacts with calf brain ribosomes in the presence of poly(U) resulting in a rapid hydrolysis of GTP and the binding of Phe-tRNA to the ribosome. Indirect evidence indicates that EF1·GDP is a product of this reaction. In the absence of poly(U) the intact complex reacts with the ribosomes without hydrolysis of GTP. The stability of the ternary complex was different with the various EF1 preparations, but the most stable complexes were prepared with calf brain EF1. Sephadex chromatography of the ternary complex shows that it contains a low molecular-weight species of the enzyme.     

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites in the tRNA.

Trans-diamminedichloroplatinum (II) was used to induce reversible crosslinks between EF-Tu and Phe-tRNA(Phe) within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex. Up to 40% of the complex was specifically converted into crosslinked species. Two crosslinking sites have been unambiguously identified. The major one encompassing nucleotides 58 to 65 is located in the 3'-part of the T-stem, and the minor one encompassing nucleotides 31 to 42 includes the anticodon loop and part of the 3'-strand of the anticodon stem.     

Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of tRNA located at the P-site on the turnover of EF-Tu.GTP on ribosomes.

The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.     

The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli

Interaction of Phe-tRNA.elongation factor Tu.GTP with poly(U)-programmed ribosomes containing an occupied P site can be described by a three-step kinetic mechanism. Initial binding is followed by the cleavage of GTP, and then a new peptide bond is formed. Rate constants controlling the first and third of these reactions are known, but only a lower limit for the rate constant of the cleavage step has been reported. We have determined this rate constant to be 20 s-1 at 5 degrees C, 30 s-1 at 15 degrees C, and 50 s-1 at 25 degrees C. This is much faster than the reverse step of the initial binding process and implies that the intrinsic accuracy of the ribosome in the initial selection step is sacrificed in favor of speed. The similarity of the kinetic and chemical mechanism of this GTP cleavage step with other nucleoside 5'-triphosphatases is discussed.     

Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.

The kinetic mechanism of elongation factor Tu (EF-Tu)-dependent binding of Phe-tRNAPhe to the A site of poly(U)-programmed Escherichia coli ribosomes has been established by pre-steady-state kinetic experiments. Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments. Initial binding to the ribosome of the ternary complex EF-Tu.GTP.Phe-tRNAPhe is rapid (k1 = 110 and 60/micromM/s at 10 and 5 mM Mg2+, 20 degreesC) and readily reversible (k-1 = 25 and 30/s). Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+-dependent manner (k-2 = 0.2 and 2/s). It induces the GTPase conformation of EF-Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg2+. The EF-Tu conformation switches from the GTP- to the GDP-bound form (k4 = 60/s), and Phe-tRNAPhe is released from EF-Tu.GDP. The accommodation of Phe-tRNAPhe in the A site (k5 = 8/s) takes place independently of EF-Tu and is followed instantaneously by peptide bond formation. The slowest step is dissociation of EF-Tu.GDP from the ribosome (k6 = 4/s). A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A-site binding.