Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome.

The fidelity of aminoacyl-tRNA (aa-tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu. Here we report the rate constants of A-site binding of a near-cognate aa-tRNA. The comparison with the data for cognate aa-tRNA reveals an additional, important contribution to aa-tRNA discrimination of conformational coupling by induced fit. It is found that two rearrangement steps that limit the chemical reactions of A-site binding, i.e. GTPase activation (preceding GTP hydrolysis) and A-site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near-cognate aa-tRNA. This suggests an induced-fit mechanism of aa-tRNA discrimination on the ribosome that operates in both initial selection and proofreading. It is proposed that the cognate codon-anticodon interaction, more efficiently than the near-cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A-site accommodation of aa-tRNA, thereby accelerating the chemical steps. As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template-programed systems in general.     

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.     

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.     

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.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes

The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments. These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H. & Fischer, E. (1982) J. Biol. Chem. 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C. The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique. It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer. A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool. Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant. According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious.     

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.     

Conformational Sampling of Aminoacyl-tRNA during Selection on the Bacterial Ribosome

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.     

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.     

Characterization of the elongation factors from calf brain. 3. Properties of the GTPase activity of EF-1 alpha and mode of action of kirromycin

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.     

Streptomycin interferes with conformational coupling between codon recognition and GTPase activation on the ribosome

Aminoacyl-tRNAs (aa-tRNAs) are selected by the ribosome through a kinetically controlled induced fit mechanism. Cognate codon recognition induces a conformational change in the decoding center and a domain closure of the 30S subunit. We studied how these global structural rearrangements are related to tRNA discrimination by using streptomycin to restrict the conformational flexibility of the 30S subunit. The antibiotic stabilized aa-tRNA on the ribosome both with a cognate and with a near-cognate codon in the A site. Streptomycin altered the rates of GTP hydrolysis by elongation factor Tu (EF-Tu) on cognate and near-cognate codons, resulting in almost identical rates of GTP hydrolysis and virtually complete loss of selectivity. These results indicate that movements within the 30S subunit at the streptomycin-binding site are essential for the coupling between base pair recognition and GTP hydrolysis, thus modulating the fidelity of aa-tRNA selection.     

The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II

A basic peptide with antiviral properties isolated from pokeweed is shown to inhibit the synthesis of globin and phenylalanine peptides on ribosomes isolated from rabbit reticulocytes. The inhibition appears to involve a specific effect of the peptide inhibitor on the larger ribosomal subunit that can be produced at a ratio of inhibitor to ribosomes of less than one to one. Ribosomes treated with the inhibitor have a reduced capacity to support enzymatic binding of Phe-tRNA to ribosomes and GTP hydrolysis caused by the elongation enzyme, EF-I. Treated ribosomes exhibit a concomitant capacity for increased GTP hydrolysis by EF-II but do not efficiently support EF-II-dependent binding of [³H]GTP. Such binding appears to involve the formation of an EF-II·GDP·ribosome complex. Thus, the inhibitor has an effect on GTP-dependent reaction carried out by both of the peptide elongation enzymes. The relation between these effects in the reticulocyte system is discussed in relation to the effects of siomycin or thiostrepton in blocking GTP hydrolysis by EF-T and EF-G on prokaryotic ribosomes.     

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.     

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.     

Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation.

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.     

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.     

Role of yeast elongation factor 3 in the elongation cycle

Investigation of the role of the polypeptide chain elongation factor 3 (EF-3) of yeast indicates that EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA (aa-tRNA) to the ribosome. In the yeast system, the binding of the ternary complex of EF-1 alpha.GTP.aa-tRNA to the ribosome is stoichiometric to the amount of EF-1 alpha. In the presence of EF-3, EF-1 alpha functions catalytically in the above mentioned reaction. The EF-3 effect is manifest in the presence of ATP, GTP, or ITP. A nonhydrolyzable analog of ATP does not replace ATP in this reaction, indicating a role of ATP hydrolysis in EF-3 function. The stimulatory effect of EF-3 is, in many respects, distinct from that of EF-1 beta. Factor 3 does not stimulate the formation of a binary complex between EF-1 alpha and GTP, nor does it stimulate the exchange of EF-1 alpha-bound GDP with free GTP. The formation of a ternary complex between EF-1 alpha.GTP.aa-tRNA is also not affected by EF-3. It appears that the only reaction of the elongation cycle that is stimulated by EF-3 is EF-1 alpha-dependent binding of aa-tRNA to the ribosome. Purified elongation factor 3, isolated from a temperature-sensitive mutant, failed to stimulate this reaction after exposure to a nonpermissive temperature. A heterologous combination of ribosomal subunits from yeast and wheat germ manifest the requirement for EF-3, dependent upon the source of the "40 S" ribosomal subunit. A combination of 40 S subunits from yeast and "60 S" from wheat germ showed the stimulatory effect of EF-3 in polyphenylalanine synthesis (Chakraburtty, K., and Kamath, A. (1988) Int. J. Biochem. 20, 581-590). However, we failed to demonstrate the effect of EF-3 in binding aa-tRNA to such a heterologous combination of the ribosomal subunits.     

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

New antibiotic that acts specifically on the GTP-bound form of elongation factor Tu.

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.