31P Magnetic relaxation studies of yeast transfer RNAPhe.

The molecular mechanism of thermal unfolding of yeast tRNA^Phe in 20 mM NaCl, 1 mM EDTA, and 10 mM MgSO₄, pH 7.1 ± 0.1, has been examined by ³¹P magnetic relaxation and the nuclear Overhauser effect methods at 40.48 MHz in the temperature range of 22.5–80°C. Two partially resolved ³¹P resonance peaks of yeast tRNA^Phe have been found to behave distinctively different in their longitudinal relaxation times. Individual intensities of the two partially resolved peaks have been quantitatively estimated by the use of relaxation data and the nuclear Overhauser effect as a function of temperature. The results of these observations largely support the earlier suggestion by Guéron and Shulman that the high- and low-field parts of the main ³¹P resonance cluster originate from phosphorus nuclei belonging to the double-helical and nonhelical regions of the tRNA, respectively. The spin-lattice relaxation of the phosphorus nucleus has been found to be determined dominantly by the dipolar interaction with the surrounding ribose protons at this observing frequency. Rotational correlation times for the two portions of the ribose-phosphate backbone of the tRNA have been separately deduced from the quantitative treatment of the ³¹P nuclear spin-lattice relaxation times (T₁) and the nuclear Overhauser effect. The result indicates that the two portions undergo internal motions at distinctively different rates of 10⁸–10¹⁰ sec^?1 order in the temperature range of 22.5–80°C, and that the thermal activation of these motions occurs at least in three distinctive steps, i.e., 22.5–31, 31–40, and 40–80°C. The rates of the internal motions and the associated activation energies in respective steps give some insight into the thermo-induced change of the yeast tRNA^Phe structure.     

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.     

Calorimetric investigations of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid

The enthalpy of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid from brewer's yeast (tRNA^Phe_{brewer's yeast}) has been measured using both an LKB 10700-2 batch miciocalorimeter and an adiabatic differential scanning calorimeter. In the mixing calorimeter the conversion from coil to helix was induced by mixing a tRNA^Phe solution with a solution containing an excess of MgSO₄. We measured the enthalpy of this reaction stepwise in the temperature range from +9 to +60° C. For the enthalpy of folding of tRNA^Phe from coil to helix this method yielded the remarkably high value of ?310 $\text{kcal}\text{mole}$ of tRNA^Phe. With the differential scanning calorimeter in which the helix-coil conversion is simply induced by raising the temperature we found a value of +240 $\text{kcal}\text{mole}$ of tRNA^Phe at a Tm value of 76° C and a value of +200 $\text{kcal}\text{mole}$ of tRNA^Phe at a T_m value of 50° C. A comparison of the apparent van't Hoff enthalpies with the calorimetrically measured enthalpies shows, that the cooperativity of the system increases continually with rising melting temperatures - which are achieved by increasing Mg²⁺ concentrations - reaching a constant value at about 57° C. Above this temperature value the thermodynamic behaviour of the helix-coil conversion of tRNA^Phe may be approximately described by the model of an all-or-none process.     

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAsPhe which differ in their minor nucleotide content

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA^Phe species, namely tRNA^Phe1 and tRNA^Phe2. By sequence determination, we show that tRNA^Phe2 is identical to the previously sequenced tRNA^Phe1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs^Phe vary during leaf development: in etiolated leaves the undermodified tRNA^Phe2 only represents 15% of total chloroplast tRNA^Phe, during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.     

Mechanism of oligonucleotide hybridization with the 3′-terminal region of the yeast tRNAPhe

Interaction of yeast phenylalanine tRNA with oligonucleotides complementary to its 3′-terminal nucleotide sequence was thoroughly studied. Using the gel retardation technique, thermodynamic and kinetic parameters of the tRNA complexation in physiological conditions were determined. Analysis of dependences of the complex formation on the oligonucleotide concentration and incubation time showed that this process proceeds in two stages. At the first stage, a metastable complex of the oligonucleotide with the open, single-stranded sequence ACCA at the 3′ end of tRNA rapidly forms. The second stage involves a slow intramolecular rearrangement of the resulting metastable complex into a full-sized heteroduplex accompanied by the tRNA^Phe unfolding. The data gained suggest that the RNA unfolding stage is limiting in the interaction of oligonucleotides with natural RNAs. Principles of selection of optimal hybridization probes and antisense oligonucleotides are discussed.     

Statistical mechanical deconvolution of thermal transitions in macromolecules. III. Application to double-stranded to single-stranded transitions of nucleic acids

The statistical mechanical deconvolution theory for macromolecular conformational transitions is extended to the case of nucleic acids transitions involving strand separation. It is demonstrated that the partition function, Q, as well as all the relevant thermodynamic quantities of the system, can be calculated from experimental scanning calorimetric data. In particular, it is shown that important thermodynamic parameters such as the size of the average cooperative unit during strand separation, the mean helical segment length, and the mean coil-segment length can be calculated from the average excess enthalpy function 〈ΔH〉. The theory is applied to the double-stranded to single-stranded transition of the system poly(A)·poly(U) at different salt concentrations. It is shown that the mean helical segment length is a monotonically decreasing function of the temperature well before strand separation occurs. On the other hand, the mean coil segment length remains practically constant until temperatures very close to T_m. Both experimental findings clearly indicate that the unfolding of poly(A)·poly(U) proceeds through the formation of many short helical sequences. The cooperative unit for the strand separation is calculated to be about 120 base pairs and essentially independent of the salt concentration. The existence of a minimum helical segment length of 10 ± 2 base pairs within the double-stranded form is calculated.     

Function of Y in codon-anticodon interaction of tRNA Phe

Molar association constants of binding oligonucleotides to the anticodon loops of (yeast) tRNA^Phe, (yeast) tRNA_HCl^Phe and (E. coli) tRNA_F^Met have been determined by equilibrium dialysis. From the temperature dependence of the molar association constants, ΔF, ΔH and ΔS of oligomer-anticodon loop interaction have been determined. The data indicate that the free energy change of codon-anticodon interaction is highly influenced by the presence of a modified purine (tRNA^Phe), of an unmodified purine (tRNA_F^Met) or its absence (tRNA_HCl^Phe). Excision of the modified purine Y in the anticodon loop of tRNA^Phe results in a conformational change of the anticodon loop, which is discussed on the basis of the corresponding changes in ΔF, ΔH and ΔS.     

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purifiedE. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNA^Glu ₂ and tRNA^Phe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNA^Glu ₂ and tRNA^Phe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion ofN ²-methylguanine was also higher, yet only in tRNA^fMet and not in tRNA^Phe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6–21 day period of cerebellar development.     

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.     

Electrostatic effects in divalent ion binding to tRNA.

The binding of Mn²⁺ to yeast tRNA^Phe at 25°C is measured by esr, and found to depend strongly on the concentration of monovalent cations, showing the importance of electrostatic effects. In low sodium (<15mM/l.), the affinity is high and the Scatchard plots are distinctly curved. In high sodium (>50mM/l.), the affinity and the curvature are reduced. In a limited range of sodium concentrations (15–30mM/l.), the folding of tRNA which is induced by the divalent ions results in cooperative binding, leading to upwards convexity of the Scatchard plot. An electrostatic model is developed, based on a single type of binding site which we take to be the phosphates, with a binding constant for Mn²⁺ in the range of that found for ApA, 10 l./M. We compute the change in the binding constant due to the electrostatic potential of the distant charges (other phosphates and counterions), using a single set of parameters for all sodium concentrations. The model predicts that the plots in low sodium are curved, and a good fit to the experimental results is obtained: it is therefore not legitimate or necessary to interpret these results in terms of two types of binding sites. In high salt, the model gives plots that are only slightly curved, corresponding to weaker electrostatic effects. This shows that a search for sites with a special binding mode should be done in high salt. The computed plots are in good agreement with the data, except for slight differences concerning the first bound ions, which give a possible indication in favor of special binding. Given the observation of one special site for Mg²⁺ at 4°C in high sodium [Stein, A. & Crothers, D. M. (1976) Biochemistry 15 , 157–160] in E. coli tRNA^fMet, we have measured the binding of Mn²⁺ at lower temperature. At 12°C, in both yeast tRNA^Phe and E. coli tRNA^fMet, the plots clearly indicate special binding. A site found in high sodium is on a very different footing from the four to six so-called strong sites unduly derived from low-salt binding plots.     

Conformational change in yeast tRNAAsp

The structure of yeast tRNA^Asp in aqueous solutions has been analyzed in the light of results obtained from Raman spectra recorded at from 5 to 82°C and compared to those of tRNA^Phe. Firm evidence is given of a reversible conformation transition for tRNA^Asp at 20°C. This transition is observed for the first time in the tRNA series. The low-temperature conformation appears to have a more regular ribose–phosphate backbone and a more effective G base-stacking. This conformational change, which occurs essentially in the D loop, could be connected to the existence of two (A and B) crystal forms obtained depending on crystallization conditions. The melting temperatures, which are different for each base stacking in tRNA^Asp, lie in a range of about 70°C, much higher than for tRNA^Phe. This fact is interpreted by a higher ratio of G-C base pairs in tRNA^Asp.     

Interaction of Complementary Oligonucleotides with the 3′-End of Yeast tRNAPHE

Abstract

Interaction of yeast tRNA^Phe with oligodeoxyribonucleotides (ONs), complementary to the nucleotides 62–76 was investigated. Results of gel-mobility shift assay and RNase A probing evidence that the ONs containing the sequence complementary to the tRNA ACCA end can easily invade the hairpin structure under physiological conditions. The limiting step of association process is the tRNA unfolding.     

High resolution NMR study of the melting of yeast tRNA Phe

The 300 MHz NMR spectra of the hydrogen bonded NH ring protons of tRNA_Yeast^Phe have been measured as a function of temperature. In the presence of Mg⁺⁺ two resonances, one from the Aψ base pair and the other probably from the neighboring base pair, disappear between 56 and 58°C. In the absence of Mg⁺⁺ the DHU stem, the acceptor stem (in particular its AU base pair #6 and #7) and the Aψ base pair in the anticodon stem melt slightly earlier than the other parts of the molecule. Since the DHU stems in tRNA_Yeast^Phe and tRNA_Coli^fMet have the same base pairing scheme it is interesting that their melting behavior is entirely different in both molecules. This is discussed in terms of the tertiary structure.     

Nucleoside Modifications Affect the Structure and Stability of the Anticodon of tRNALys,3

Abstract

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNA^Lys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNA^Phe and provides an explanation of the critical role of modification in this tRNA.     

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.     

Solvent accessibility study on tRNAPhe

In order to assess the solvent–solute association in the tRNA^Phe molecule, solvent accessibility calculations were carried out for its crystalline and completely extended states following the method of Lee and Richards. To do this, results from the calculations on model trinucleotide systems pApXpA with different bases at position X were used. In the folded form of the molecule, it was found that the oxygen atoms O(I) and O(II) of almost all the phosphate groups and the O(2′) atoms of the sugar rings situated throughout the backbone were highly exposed to the solvent. The amount of reduction found in the solvent accessibilities of the various atoms in going from the extended state to the folded state of the molecule indicates the kind of compactness of the tertiary structure in tRNA^Phe. The results give quantitative support to many characteristics of the tRNA molecule, such as loop sections, buried/exposed residues, hydrophobic interactions, etc., which were thought to be due to other factors.     

Crystallization of tRNAs as Cetyltrimethylammonium Salts

VARIOUS species of transfer RNA have been crystallized by controlled precipitation from aqueous solutions containing organic solvents or ammonium sulphate (reviewed in refs. 1 and 2). These methods have produced a great variety of crystal forms which, with a few exceptions^3,4, are usually poorly ordered as judged by X-ray diffraction. This is probably because the interactions between molecules are few and rather nonspecific, making the crystal structure extremely sensitive to the crystallization conditions. For this reason, attempts have been made to crystallize tRNA as the cetyltrimethylammonium (CTA-) salt. The additional interaction between hydrophobic cetyl cations bound to the different molecules may stabilize the crystal lattice and have a positive effect on the crystallization process and therefore on the order of the crystals. We report here the production of crystals of CTA-salts of five different tRNAs; tRNA^Met_f, tRNA^Glu, tRNA^Phe, tRNA^Tyr from E. coli and tRNA^Phe from yeast. In the case of tRNA^Met_f, different crystal forms were obtained in the presence of different cations.     

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.