The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA^Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA^Tyr complex with [³H]acetic anhydride. In a second step the two ³H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [¹⁴C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ${}^{14}\text{C}{}^3\text{H}$ ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNA^Tyr was suggested from altered ${}^{14}\text{C}{}^3\text{H}$ ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA^Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA^Tyr complex has been detected.Only one molecule of tRNA^Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.     

Mechanism of suppression in Drosophila: a change in tyrosine transfer RNA

The mechanism of suppression of the vermilion locus in Drosophila melanogaster is examined. The suppressor locus, su(s)², is shown to control directly the amount of a specific tyrosine transfer RNA which occurs in the adult fly. Wild-type flies have three chromatographic forms of tyrosine tRNA but flies that are homozygous for the suppressor gene su(s)² contain little or none of the second chromatographic form. The isoacceptor patterns of tRNA for leucine, phenylalanine and serine are identical in the suppressor mutant and wild-type fly. Genetic data show that the phenotypic expression of su(s)² and the altered chromatographic pattern of tyrosine tRNA are recessive and that both map at the same position on the left tip of the X chromosome. Furthermore, another suppressor of vermilion was induced by ethyl methane sulfate, su(s)^e1, that is at the same locus as su(s)² and that produces the same change in tyrosine tRNA as su(s)².     

A role for ribonuclease III in synthesis of bacteriophage T4 transfer RNAs.

Synthesis of T4 tRNA^Gln depends on normal levels of $\text{Escherichia}\text{coli}$ ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA^Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA^Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA^Gln.     

Differences in aminoacylation in vivo and in vitro of lysine isoaccepting tRNAs from virus-transformed cells

When murine sarcoma virus-transformed cells are labeled with [³H]lysine $\text{in}\text{vivo}$ for various periods, 5 of 6 isoaccepting lysine tRNAs separable by RPC-5 chromatography are aminoacylated in 1 hr to the same extent that they are aminoacylated $\text{in}\text{vitro}$. The sixth isoacceptor, tRNA₆^Lys, is not aminoacylated $\text{in}\text{vivo}$ to a measurable extent in 1 hr, although it is present in the tRNA prepared from the cells. All six isoacceptors are aminoacylated with [³H]lysine $\text{in}\text{vivo}$ when the labeling period is 2 or 3 hr. These results further show that $\text{in}\text{vitro}$ correlations of the amount of tRNA₄^Lys with cell division accurately reflect the situation $\text{in}\text{vivo}$. Results of differential centrifugation indicate that tRNA₆^Lys occurs in mitochondria.     

A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

Tyrosyl-tRNA synthetase catalyzes the attachment of tyrosine to the 3′ end of tRNA^Tyr, releasing AMP, pyrophosphate, and l-tyrosyl-tRNA as products. Because this enzyme plays a central role in protein synthesis, it has garnered attention as a potential target for the development of novel antimicrobial agents. Although high-throughput assays that monitor tyrosyl-tRNA synthetase activity have been described, these assays generally use stoichiometric amounts of tRNA, limiting their sensitivity and increasing their cost. Here, we describe an alternate approach in which the Tyr-tRNA product is cleaved, regenerating the free tRNA substrate. We show that cyclodityrosine synthase from Mycobacterium tuberculosis can be used to cleave the l-Tyr-tRNA product, regenerating the tRNA^Tyr substrate. Because tyrosyl-tRNA synthetase can use both l- and d-tyrosine as substrates, we replaced the cyclodityrosine synthase in the assay with d-tyrosyl-tRNA deacylase, which cleaves d-Tyr-tRNA. This substitution allowed us to use the tyrosyl-tRNA synthetase assay to monitor the aminoacylation of tRNA^Tyr by d-tyrosine. Furthermore, by making Tyr-tRNA cleavage the rate-limiting step, we are able to use the assay to monitor the activities of cyclodityrosine synthetase and d-tyrosyl-tRNA deacylase. Specific methods to extend the tyrosyl-tRNA synthetase assay to monitor both the aminoacylation and post-transfer editing activities in other aminoacyl-tRNA synthetases are discussed.     

The antisuppressor strain sin1 of Schizosaccharomyces pombe lacks the modification isopentenyladenosine in transfer RNA

The modified base N⁶-(Δ2-isopentenyl)-adenosine (i⁶A) is missing in all transfer RNAs isolated from the antisuppressor strain sin1 of Schizosaccharomyces pombe. i⁶A is found adjacent to the 3′ side of the anticodon of several tRNAs of S. pombe. Sequence analysis of tyrosine tRNA from the antisuppressor strain sin1 shows an unmodified adenosine instead of the i⁶A. i⁶A-deficient tyrosine tRNA elutes much earlier than wild-type tRNA^Tyr during reverse phase chromatography (RPC-5). Serine tRNA and tryotophan tRNA from the sin1 mutant show a similar shift in the elution profile. We therefore conclude that these two tRNAs are also deficient in i⁶A. The presence of the antisuppressor mutant sin1 leads to inactivation of the nonsense suppressor sup3-i. As sup3-i is a mutated serine tRNA, we conclude that the loss of the modification i⁶A on the suppressor tRNA is responsible for the inactivation of sup3-i. Compared to wild type, the growth rate of the sin1 strain is only slightly reduced and the other i⁶A-deficient tRNAs seem to function normally. We assume that the sin1 mutation affects the structural gene of an enzyme in the isopentenyl pathway, probably the transferase.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

The effect of the Q base modification on the usage of tRNAHis in globin synthesis

The incorporation of histidine by two competing histidine isoaccepting tRNA species into rabbit globin in a rabbit reticulocyte lysate was studied. The results show that incorporation by each isoacceptor is in proportion to its abundance, indicating that neither species is used preferentially. In a previous study (McNamara and Smith (1978) J. Biol. Chem. $\text{253}$, 5964–5970) we showed that neither tRNA^His species responds preferentially to either of the histidine codons and that there is no preferential incorporation by either species into any histidine-containing site in either globin subunit. The Q base modification is found in one tRNA^His isoacceptor while the other is hypomodified in this this characteristic. The results indicate that none of the aspects of tRNA function in translation that have been examined is affected by Q base.     

The genes coding for tRNATyr of Drosophila melanogaster: Localization and determination of the gene numbers

Transfer RNA^Tyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNA^Tyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA^Tyr genes. The regions 22F and 85A each contain four to five tRNA^Tyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA^Tyr genes.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.     

Temperature sensitive mutants of Escherichia coli for tRNA synthesis

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3⁺-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA^Tyr was demonstrated. Electrophoretic fractionation of ³²P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions.     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.