New chromatographic and biochemical strategies for quick preparative isolation of tRNA

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA^Phe from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA^Ile from E.coli. Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA^Ile is recovered with high specific activity. (iii) tRNA_i^Met from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.coli. The tRNA_i^Met recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.     

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Two fractions of phenylalanine tRNA (tRNA^Phe₁ and tRNA^Phe₂) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNA^Phe₂ showed its conversion into more stable tRNA^Phe₁, suggesting that the two fractions have essentially the same primary structure. Both tRNA^Phe₁ and tRNA^Phe₂ had about the same acceptor activity, but tRNA^Phe₂ was aminoacylated much faster than tRNA^Phe₁. RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNA^Phe₂ than of phe-tRNA^Phe₁ but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNA^Phe from barley embryos suggest that it contains Y base similar to Y_w from wheat tRNA^Phe.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met⁻, Cys⁻, relA⁻ mutant, was previously shown to contain an altered tRNA^Ile which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNA^Ile and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA^Ile purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA^Ile (from cysteine-starved cells) was found to have a 5-fold increased V_max in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA^Ile bound more efficiently to its synthetase compared to normal tRNA^Ile. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA^Ile contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA^Ile contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA^Ile without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA^Ile has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA^Ile from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA^Ile for aminoacylation is discussed.     

Half molecules of serine-specific transfer ribonucleic acids from yeast

The preparation and analysis of half molecules from tRNA^Ser are described. Two pG-halves were isolated which differed only in the presence or absence of an acetyl group on the cytidylic acid residue at position 12. The CCA-half derived from tRNA₁^Ser was isolated pure, while the CCA-half derived from tRNA₂^Ser was isolated as a mixture with the CCA-half from tRNA₁^Ser from which the terminal CpCpA had been cleaved off.The acceptor activity of the combined complementary half molecules was 90% of the one of intact tRNA^Ser. The Michaelis constant and maximal velocity of amino-acylation were found to be identical for tRNA^Ser and the combined fragments.When half molecules were present at different ratios in aminoacylation studies it was found that one pG-half molecule can mediate the charging of several CCA-half molecules. There are indications that the CCA-half molecule alone can accept some serine. The CCA-half molecule alone can be aminoacylated to a rather high degree in the presence of an excess of tRNA_ox^Ser or tRNA^Ser-a and to a small degree in the presence of tRNA_ox^Ala (yeast) but not at all in the presence of tRNA_ox^Phe or tRNA_ox^Val (E. coli).Combinations of half molecules from tRNA^Ser with the opposite half molecules from tRNA^Phe could not be aminoacylated with Ser or Phe or 15 other amino acids although one of the combinations was well associated according to gel electrophoresis and differential melting curves.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNA^Phe and tRNA_init, Escherichia coli tRNA_init and HIV tRNA^Lys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem. 2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg²⁺. Results showed a stabilizing effect associated with the presence of the modifications and Mg²⁺ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNA_init and limitations were observed in the conformation of E. coli tRNA_init, the presence of the modified nucleotides and of Mg²⁺ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.     

Conformational changes in the thiouridine region of Escherichia coli transfer RNA as assessed by photochemically induced crosslinking: Effect of a single base change in the “extra” loop of formylmethionine tRNA and of denaturation of tryptophan tRNA

Photochemical crosslinking studies on two formylmethionine tRNAs of Escherichia coli are consistent with the hypothesis that the role of 7-methylguanosine is to stabilize a tertiary structure of tRNA in which the “extra” loop is folded over so as to be close to the 4-thiouridine region of the molecule. In tRNA^{fmet 3}, which differs from tRNA^{fmet 1} only by substitution of an adenosine for the 7-methylguanosine in the “extra” loop, crosslinking was virtually abolished when the tRNA was placed in 40 mm Na⁺, whereas tRNA^{fmet 1} in 40 mm Na⁺ was crosslinked to 95% of the maximum extent observed for both tRNAs in Mg²⁺. Even in Mg²⁺, a difference in structure between the two tRNAs could be detected by means of a two-fold decrease in the rate of crosslinking in tRNA^{fmet 3} as compared to tRNA^{fmet 1}. Comparison of crosslinking in the native and metastable denatured forms of tRNA^Trp of E. coli revealed that these structures also differ with respect to the orientation and/or distance between 4-thiouridine (8) and cytidine (13), since denaturation abolished crosslinking. However, separation of these two residues is not obligatory for denaturation, since crosslinked tRNA^Trp could still be denatured. A 30% difference in fluorescence between the native and denatured forms of crosslinked-reduced tRNA^Trp infers an increase in hydrophilicity in the 4-thiouridine region upon denaturation.     

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNA^Phe, Glu-tRNA^Glu and Met-tRNA_m^Met from Escherichia coli and Phe-tRNA^Phe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA₂^Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA₂^Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.     

Conformational peculiarities of rRNA inff supMet from E. coli as revealed by fluorescent methods

Conformational transitions in several individual tRNAs (tRNA _inff ^supMet , tRNA^Phe from E. coli, tRNA _inf1 ^supVal , tRNA^Ser, tRNA^Phe from yeast) have been studied under various environmental conditions. The binding isotherms studies for dyes-tRNA complexes exhibited similarities in conformational states of all tRNAs investigated at low ionic strength (0.01 M NaCl). By contrast, at high ionic strength (0.4 M NaCl or 2×10^-4 M Mg²⁺) a marked difference is found in structural features of tRNA _inff ^supMet as compared with other tRNAs used. The tRNA _inff ^supMet is the only tRNA species that does not reveal the strong type of complexes with ethidium bromide, acriflavine and acridine orange.     

Protein synthetic ability of Escherichia coli valine transfer RNA with pseudouridine, ribothymidine, and other uridine-derived residues replaced by 5-fluorouridine

The requirement for pseudouridine and other uridine-derived minor nucleotides for activity of transfer RNA in several of the intermediate steps in protein synthesis was examined using a purified preparation of Escherichia coli valine transfer RNA in which the uridine and uridine-derived nucleotides were replaced by 5-fluorouridine. The degree of substitution was 87% or better for uridine, pseudouridine, ribothymidine, dihydrouridine, and 4-thiouridine, and at least 75% for uridine-5-oxyacetic acid. Each of these nucleotides, except for uridine, occurs only once in this transfer RNA species.The rate and yield of ternary complex formation with elongation factor Tu-GTP of E. coli, the rate and extent of elongation factor-dependent binding to ribosomes at 10 mm-Mg²⁺, and the rate and extent of synthesis of the co-polypeptide (Phe_n,Val) dependent on poly(U₃,G) were all unchanged when the fluorouridine-containing transfer RNA was used in place of the normal control. In all yield assays, the amount of product formed was proportional to the amount of valyl-tRNA added. Non-enzymatic binding to ribosomes in the presence of tetracycline was more efficient for the fluorouridine-substituted tRNA than for the control. At 15 to 20 mm-Mg²⁺ the polynucleotide-dependent binding, as a percentage of tRNA added, was 44% for the control and 65% for the modified tRNA, while at 5 mm-Mg²⁺, the figures were 10% and 40%, respectively.We conclude from these results that there is no essential requirement for pseudouridine or ribothymidine in the GTψC loop of tRNA for its proper functioning in protein synthesis in vitro. Confirming earlier work, dihydrouridine and 4-thiouridine are also not essential.     

Complex formation between transfer RNAs with complementary anticodons: use of matrix bound tRNA

It is shown that yeast tRNA^Phe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNA^Phe in its ability to form a specific complex with $\text{E. coli}$ tRNA₂^Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpA_OH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N⁷-methylguanine at position 46 (m⁷G46) in tRNA is produced by tRNA (m⁷G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m⁷G46 modification in tRNA^Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m⁷G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m¹G37, suggesting that the m⁷G46 positively affects their formations. Although the lack of the m⁷G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA^Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA^Phe and tRNA^Ile. ³⁵S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m⁷G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m⁷G46 modification supports introduction of other modifications.     

Changes in in vivo levels of charged transfer RNA species during development of the posterior silkgland of Bombyx mori

The changes in the in vivo levels of acylated tRNA specific for the amino acids that make the bulk of silf-fibroin were examined during the distinct physiological phases, the growth phase and the silk-fibroin production phase, of the developing posterior silkgland of Bombyx mori. The levels of tRNA acylated with glycine and alanine, the major amino acid components of silk-fibroin, increased about 30-fold during the transition from growth stage to fibroin production stage in each gland, whereas the increments in the levels of other aminoacylated tRNA's were substantially low. An analysis of the iso-accepting species of tRNA^Gly, the most abundant tRNA in silkgland, on benzoylated DEAE-cellulose columns showed that the levels of acylated tRNA^Gly species increased differentially during the same stages of development. These results suggested that the quantitative alterations in aminoacylated tRNA population were closely associated with the changes in protein synthesis during the terminal differentiation of silkgland.