Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

Purification and properties of a T4 bacteriophage factor that modifies valyl-tRNA synthetase of Escherichia coli.

After T4 bacteriophage infects Escherichia coli, a peptide tau, produced under the control of a phage gene, binds to the host valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) and thereby changes several of its physicochemical properties. The interaction of tau with the host enzyme was investigated in vitro after extensively purifying the factor from T4-infected E. coli using a rapid purification procedure. The tau preparation migrated as a single, protein-staining band with a molecular weight of 11,000 during sodium dodecyl sulfate-gel electrophoresis. The purified peptide completely converted partially purified valyl-tRNA synthetase from uninfected E. coli into the form present in cell-free extracts prepared from virus-infected bacteria. The enzyme modified in vitro also exhibited the enhanced affinity for tRNA characteristic of the viral form of valyl-tRNA synthetase. The addition of bulk tRNA from E. coli B, tRNAVal, or tRNA1Val to enzyme modified in vitro increased its sedimentation rate to that of enzyme prepared from phage-infected cells. Amino acid analysis of the purified tau peptide revealed a relatively high concentration of the amino acids lysine and alanine, and a lack of detectable proline, tyrosine, phenylalanine, and methionine.     

Effects of magnesium and ionic strength on the diffusion and charge properties of several single tRNA species.

The technique of laser light scattering was used to evaluate the effects of Mg+2 and ionic strength on the solution structures of seven tRNA species. Information about ion effects on both conformation and electric charge were derived from measurements of the translational diffusion constants and diffusive virial coefficients. E. coli tRNAMetf and six elongator tRNAs from both Class I and II were studied. The diffusion measurements show that the responses of all but the initiator species are qualitatively similar to each other and to that of bulk tRNA, but that significant quantitative differences also obtain. All of the elongator species exhibited an anomolous increase in diffusivity reported earlier by us for bulk tRNA when placed in a low salt-low Mg+2 condition. The initiator tRNA did not undergo this transition and unlike the other tRNAs tested was apparently more compact in 1 mM Mg+2 than 10 mM Mg+2 at ionic strengths in excess of 0.1 M. At 0.1 M ionic strength, pH 7.2, the average net charge of the tRNAs ranged from 7-12 e- in 1 mM Mg+2 and 3-7 e- in 10 mM Mg+2, consistent with the binding of 1-2 additional Mg+2 ions in the higher Mg+2 condition.     

Rabbit liver tRNA1Val:II. unusual secondary structure of T psi C stem and loop due to a U54:A60 base pair.

In contrast to all other known tRNAs, mammalian tRNA1Val contains two adenosines A59 and A60, opposite to U54 and psi 55 in the U psi CG sequence of the T psi C loop, which could form unusual A:U (or A: psi pairs in addition to the five "normal" G:C pairs. In order to measure the number of G:C and A:U (A: psi) pairs in the T psi C stem, we prepared the 30 nucleotide long 3'-terminal fragment of this tRNA by "m7G-cleavage". From differentiated melting curves and temperature jump experiments it was concluded that the T psi C stem in this fragment is in fact extended by an additional A60:U54 pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the tRNA1Val fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U54 is far less available for enzymatic methylation in mammalian tRNA1Val compared to tRNA from T-E. coli. This clear difference in U54 reactivity, together with the identification of an extra A60:U54 pair in the U psi CG containing fragment suggests the presence of a 6 base pair T psi C stem and a 5 nucleotide T psi C loop in this tRNA.     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.     

The primary structure of tRNA Val 2a from baker's yeast]

The primary structure of tRNAVal2a from baker's yeast has been determined. The general methods of the investigation are presented. Twenty six distinguished points can be noted in the tRNAVal2a and tRNA1Val from baker's yeast. The anticodon region of tRNAVal2a is represented by the sequence NAC, where N corresponds to a uridine analogue nucleoside of unknown structure. The comparison of primary structures of tRNAVal2a, tRNAVal2a, tRNA1Val from E. coli and tRNAVal2a and tRNA1Val from baker's yeast is analysed.     

The composition and properties of the Escherichia coli 5-S RNA-protein complex

At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg Cl2, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possess properties relevant to the function of the 50-S subunit; it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x 10(-7) M. The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits. The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichia coli ribosome.     

Conformations of the core nucleosome: effects of ionic strength and high mobility group protein 14 and 17 binding on the fluorescence emission and polarization of dansylated methionine-84 of histone H4

Chicken histone H4 labeled at Met-84 with the fluor N-[(acetylamino)ethyl]-8-naphthyl-amine-1-sulfonic acid has been incorporated into a nucleosome which has physical characteristics virtually identical with those of native core nucleosomes. The fluorescence emission and polarization properties of the labeled nucleosome were measured as a function of ionic strength and the binding of high mobility group (HMG) proteins 14 and 17. Also, the accessibility of the fluor to the quenching agent acrylamide was determined. It was found that the fluorescence emission changes in the range 0.1-1000 mM NaCl are rather small and indicate that no major unfolding of the octamer structure occurs around Met-84 on H4 at least. Five or perhaps six discrete states were found in that ionic strength range. Each has a different accessibility to the quenching agent. The range of accessibilities varied from 9 X 10(-7) to 32 X 10(-7) mol-1 s-1 for 0.1-1000 mM NaCl, respectively. Polarization measurements showed that there was little change in the rotational relaxation lifetime of the fluor at ionic strengths less than 50 mM NaCl. Above this value, the rotational relaxation lifetimes decreased from 107 to 25 ns at 600 mM NaCl, indicating a moderately increased rotational freedom for the fluor. It is suggested that the histone octamer changes its degree of compaction in the range 0.1-600 mM NaCl but that no major protein unfolding occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.     

Effect of temperature and ionic strength on the dissociation kinetics and lifetime of PNA-DNA triplexes

Dissociation kinetics of triplexes formed by molecules of peptide nucleic acid (PNA) and DNA have been studied. The complexes consisted of oligomeric PNA containing 10 thymine bases and the dA(10) target incorporated in single-stranded (ssDNA) or double-stranded DNA (dsDNA). Their dissociation was followed by means of the gel mobility shift assay at various temperatures and sodium ion concentrations. In all experiments, the dissociation kinetics of triplexes were exponential; the effective lifetime of a triplex, tau, depended on temperature in accordance with the Arrhenius law. The tau values for T(10) PNA complexes with ss- and dsDNA were equal within the accuracy of experiments. The activation energy, U, value for T(10) PNA-DNA complexes did not change when the NaCl concentration was increased from 50 to 200 or 600 mM. Conversely, the tau values decreased with the increase in NaCl concentration. The equal lifetimes of the T(10) PNA-DNA triplexes containing ss- and dsDNA suggest that the loop formed in dsDNA does not noticeably affect the triplex structure. The decrease in the triplex lifetime tau with an increase in ionic strength was accounted for by the fact that the PNA backbone is neutral. The lack of relationship between the activation energy of dissociation and salt concentration suggests that the dissociation enthalpy does not depend on the ionic strength. Thus, the effect of ionic strength on the lifetime is entropic by its nature. Contrary to this, for complexes of ssDNA with bis-PNA 1743, which also consists of 10 thymine bases but contains 2 additional positive charges inside the sequence in 1 of the PNA arms, an increase of the dissociation enthalpy at low salt concentration was observed. We suggest that this effect is a result of a direct electrostatic interaction of the positive charges of the PNA with the DNA backbone. Finally, our results allow an estimate of the lifetime of a 10-mer triplex invasion complex in dsDNA at 37 degrees C in excess of several hundred days.     

The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase

Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT). The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA. The in vitro synthesized 17mer T-arm of E. coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT. The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA. The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold. The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA.     

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.     

N2-guanine specific transfer RNA methyltransferase I from rat liver and leukemic rat spleen

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Salt effects on internal motions of superhelical and linear pUC8 DNA. Dynamic light scattering studies

The plasmid pUC8 (2717 bp) has been studied in its native superhelical and Eco RI-linearized forms by dynamic light scattering at NaCl concentrations from 1.1 mM to 1 M. The data were analyzed using the biexponential model for the dynamic structure factor described by us in a previous paper (J. Langowski, U. Giesen and C. Lehmann, Biophys. Chem. 25 (1986) 191). As before, we could identify two decay components corresponding to the center-of-mass diffusion and to internal motions of the DNA, where the fast component could be identified as a rotational diffusion contribution in the case for superhelical, but not for linear DNA. We found that the conformation of superhelical pUC8 is not affected by changing the ionic strength, while the amplitude of the internal relaxation increases approx. 2-fold when [NaCl] is raised from 1.1 mM to 1 M. The linearized DNA shows an increase of the diffusion coefficient with ionic strength which is, however, not quite as pronounced as that found by others (Z. Kam, N. Borochov and H. Eisenberg, Biopolymers 20 (1981) 2671), and, together with the unchanged conformation of the superhelical DNA, suggests a persistence length which is not strongly dependent on ionic strength. In contrast to the increasing amplitude of internal relaxation for the superhelical DNA, this amplitude remains constant or decreases slightly for linear DNA on going from 1.1 mM to 1 M salt. Our findings are further discussed with respect to possible models of the interwound form of superhelical DNA.     

Separation and purification of periplasmic cytochrome c553 using reversed micelles

Surfactant concentration, ionic strength, and pH were optimised for the selective separation and purification of periplasmic cytochrome c553 from recombinant E. coli TG2 cells using response surface methodology. Back-extraction was accomplished using counter-ionic surfactant addition. Optimum forward extraction conditions were: 65 mM bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), 0.07 M NaCl, and pH 8.4, while the optimum back-extraction conditions were 80 mM trioctylmethylammonium chloride, 0.85 M KCl, and pH 9.62. In comparison to a conventionally purified sample using column chromatography (10 mg cytochrome c553 l–1 with a purity of 0.66), reverse micelles achieved the same concentration and similar purity (0.50) in only two simple steps.     

Derepression of alkaline phosphatase in Escherichia coli by p-fluorophenylalanine

p-Fluorophenylalanine (FPA) causes a 100-fold increase in alkaline phosphatase in Escherichia coli B, strain PR1 at 30 C in minimal medium that contains excess inorganic phosphate (1.92 x 10(-3)m). Little increase in alkaline phosphatase synthesis occurs under these conditions at 22 C. [This strain is known to have a mutation in a regulator gene (R(2)) that, in the absence of FPA, permits derepression of alkaline phosphatase synthesis at 37 C, but not at 30 C or below.] In contrast, E. coli B3 (the strain from which E. coli B strain PR1 was derived) is not derepressed at 30 C by FPA. (14)C-FPA is incorporated into bacterial proteins. Temperature-shift experiments (30 Cright harpoon over left harpoon22 C) in the presence of FPA are consistent with the following mechanism. FPA is incorporated into the genetically altered R(2) protein at 30 and 22 C. This further alteration due to the incorporation of analogue makes the R(2) protein inactive at 30 C, but active at 22 C.     

Purification and characterization of a mutant tRNA nucleotidyltransferase

tRNA nucleotidyltransferase has been extensively purified from a mutant strain of Escherichia coli which displays greatly decreased AMP incorporation, but normal CMP incorporation. The defect in AMP incorporation is retained throughout the purification of the mutant protein. The mutant protein behaves identically to the wild-type protein with regard to elution position on various chromatographic columns, and both have similar molecular weights of about 50000. The defect in the mutant protein is accentuated by the use of yeast tRNA rather than E. coli tRNA-C--C as substrate, by decreased pH, by increased ionic strength and by decreased divalent cation concentration. Substitution of MN2+ for Mg2+ greatly increases the relative activity of the mutant enzyme. In all these cases, CMP incorporation by the mutant enzyme remains the same as the wild-type enzyme. The Km values of the mutant enzyme for its tRNA and triphosphate substrates are unchanged, and the mutant protein is as stable as the wild type with respect to temperature inactivation. These results strongly suggest that the mutation is in the structural gene for tRNA nucleotidyltransferase, and that the mutation probably does not affect the overall structure of the mutant protein, but only a localized region near the AMP-incorporating site.     

Multidimensional diffusion modes and collision frequencies of cytochrome c with its redox partners

We have determined the modes and rates of cytochrome c diffusion as well as the collision frequencies of cytochrome c with its redox partners at the surface of the isolated, mitochondrial inner membrane over a broad range (0-150 mM) of ionic strengths. Using fluorescence recovery after photobleaching, resonance energy transfer, and direct binding assay, we determined that the diffusion coefficient of cytochrome c is independent of its concentration and quantity bound to the inner membrane, that the distance of cytochrome c from the membrane surface increases with increasing ionic strength, and that there is no significant immobile fraction of cytochrome c on the membrane regardless of ionic strength. The rate of cytochrome c diffusion increases while its mode of diffusion changes progressively from lateral to three-dimensional with increasing ionic strength. At physiological ionic strength (100-150 mM), the diffusion of cytochrome c is three-dimensional with respect to the surface of the inner membrane with a coefficient of 1.0 x 10(-6) cm2/s, and little, if any cytochrome c is bound to the membrane regardless of its concentration. Furthermore, as ionic strength is raised from zero to 150 mM, the cytochrome ckd for the inner membrane increases, its mean occupancy time on the inner membrane to collide with a redox partner (tau) decreases, and its diffusion-based collision frequencies with its redox partners decrease. These data reveal the significance of both diffusion and concentration (affinity) of cytochrome c near the surface of the inner membrane in the control of the collision frequency of cytochrome c with its redox partners.     

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.