Proton exchange rates in transfer RNA as a function of spermidine and magnesium

Solvent exchange rates of selected protons were measured by NMR saturation recovery for E. coli tRNAVal, E. colifMet and yeast tRNAPhe, at temperatures from 20 to 40 degrees C, in the presence of 0.12M Na+ and various levels of added spermidine. tRNAVal was also studied with added Mg++. The exchange rates in zero spermidine and Mg++ indicate early melting of the U8 A14 interaction, in accord with thermodynamic melting studies. Exchange rates for secondary protons suggest early melting of the T stem in tRNAfMet and the acceptor stem in tRNAPhe, in contradiction with melting transition assignments from thermodynamic work. Addition of 10 spermidines per tRNA stabilizes the secondary and tertiary interactions more effectively than added Na+, but less so than Mg++. Added spermidine has the curious effect of increasing the exchange rate of the psi 55 N1 proton, while protecting the psi 55 N3 proton from exchange in all three tRNA's. Added Mg++ has the same effect on tRNAVal.     

Proton nuclear magnetic resonance study of the effect of pH on tRNA structure.

The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6.     

An NMR study of the exchange rates for protons involved in the secondary and tertiary structure of yeast tRNA Phe.

Solvent exchange rates of all the protons of yeast tRNAphe resonating in the lowfield NMR region (-11 to-15 ppm from DSS) have been measured by saturation-recovery long-pulse Fourier transform NMR. All these protons in yeast tRNAphe are in the fast exchange limit with H2O relative to their intrinsic longitudinal relaxation processes. Most rates show very little temperature dependence; however, tertiary base pair protons are preferentially destabilized in the absence of Mg++ at higher temperatures. The measured exchange rates are between 2 and 125 sec-1 for a temperature range from 10 degrees C to 45 degrees C and MgCl2 concentrations between 0 and 15 mM.     

A new method to monitor the rate of conformational transitions in RNA.

Many RNAs need Mg2+to produce stable tertiary structures. Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+dependence. Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds. We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNAPhe) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique. We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNAPhe measured previously by temperature-jump relaxation kinetics. Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.     

15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR

The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum.     

Thermal and Mg2+ dependent behavior of pseudouridines at 39th and 55th of yeast tRNAPhe

Pseudouridine psi 55 alone and both psi 55 and psi 39 in yeast tRNAPhe are selectively modified with fluorescent reagent of 4-bromomethyl-7-methoxycoumarin (BMC). The change of fluorescence intensity was measured as a function of temperature and Mg2+ concentration. Fluorescent quenching shows the stacked and unstacked forms of Y base, dependent on Mg2+ concentration. In contrast, Mg2+ had no effect on psi 55-BMC in T psi C loop at 20 degrees C. Fluorescence on titrating Mg2+ exhibited a kind of Mg2+-induced structural collapse at the corner of L-structure. The melting of psi 55-BMC takes place at 70 degrees C in 10mM Mg2+. At very low Mg2+ concentration, melting takes place at 35 degrees C. The melting of psi 39-BMC, located near the anticodon loop, was observed before the unfolding of the whole structure of tRNAPhe. A conformational transition of the anticodon loop takes place at a lower temperature and it is also expected in the quenching experiment of Y base.     

Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum.

A comparison of imino proton NMR spectra of yeast tRNAPhe recorded at various solution conditions indicates, that polyamines have a limited effect on the structure of this tRNA molecule. Polyamines are found to catalyse the solvent exchange of several imino protons in yeast tRNAPhe not only of non hydrogen bonded imino protons, but also of imino protons of the GU and of some AU and tertiary base pairs. It is concluded that at low levels of catalysing components the exchange rates of the latter protons are not determined by the base pair lifetime. In the presence of high levels of spermidine the solvent exchange rates of imino protons of several base pairs in the molecule were assessed as a function of the temperature. Apparent activation energies derived from these rates were found to be less than 80 kJ/mol, which is indicative for (transient) independent opening of the corresponding base pairs. In the acceptor helix the GU base pair acts as a dynamic dislocation. The AU base pairs at one side of the GU base pair exhibit faster transient opening than the GC base pairs on the other side of this wobble pair. The base pairs m2GC10 and GC11 from the D stem and GC28 from the anticodon stem show relatively slow opening up to high temperatures. Model studies suggest that 1-methyladenosine, an element of tRNA itself, catalyses imino proton solvent exchange in a way similar to polyamines.     

Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs

An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestions at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5S rRNA from E. coli, Thermoplasma acidophilum and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA are discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.     

Poly(U)-dependent interaction of yeast tRNA(Phe) and its fragments with ribosomes from Escherichia coli

The poly(U)-dependent bindings of yeast tRNAPhe, its derivative depleted of 3'-terminal adenosine, and 15-nucleotide having a sequence of yeast tRNAPhe anticodon arm to the P site of Escherichia coli 70S ribosomes were compared. The equilibrium and rate constants were determined. Data indicate that the anticodon arm (N28-N42) contributes the major fraction of the binding free energy (-45.3 kJ/mol at 10 mM Mg2+ and 30 degrees C). Other parts of the tRNAPhe molecule besides A76 (N1-N27 and N43-N75) bring additional-6.0 kJ/mol, and A76 contributes-2.4 kJ/mol.     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

A spin label study of the thermal unfolding of secondary and tertiary structure in E. colic transfer RNAs.

The molecular mechanism of thermal unfolding of E. coli tRNAGlu, tRNAfMet and tRNAPhe (in 0.02M Tris-HC1, pH 7.5. 10 MM Mg C12) has been examined by the spin-labeling technique. The rate of tumbling of the spin label has been measured as a function of temperature for ten different selectively spin-labeled tRNAs. Only spin labels at position s4U-8 were able to probe the tertiary structure. Evidences are presented which support the hypothesis that the thermal denaturation of the three species of tRNAs studied is sequential. The unfolding process occurs in three discrete stages. The first step (30 degrees-32 degrees) could either be assigned to a localized reorganization of the cold-denatured structure or to a "transient" melting, followed by the simultaneous disruption of the tertiary structure and part of the hU helix. This transition is observed even in the absence of magnesium. The second step (50 degrees-54 degrees) involves the melting of the anticodon and miniloop regions. The last step occurs above 65 degrees where the t psi c and amino acid acceptor stems, forming one continuous double helix, melt. A simple dynamic model is considered for tRNA function in protein biosynthesis.     

15N-labeled Escherichia coli tRNAfMet, tRNAGlu, tRNATyr, and tRNAPhe. Double resonance and two-dimensional NMR of N1-labeled pseudouridine

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.     

Interactions of some naturally occurring cations with phenylalanine and initiator tRNA from yeast as reflected by their thermal stability

The thermal unfolding of phenylalanine and initiator tRNA from yeast was investigated over a broad range of solution conditions by differential ultraviolet absorption at 260 nm. Under most conditions, the initiator tRNA exhibits two clearly separated transitions in its differential melting curve which were assigned to unfolding of tertiary and secondary structure elements, respectively. The tertiary transition of this tRNA and the overall transition observed for tRNAPhe do not show a maximum in a curve of Tm values plotted as a function of [Na+]. Such a maximum is usually observed for other nucleic acids at about 1 M Na+. In the presence of 5 mM of the divalent cation Mg2+ (or Ca2+), an overall destabilization of the tRNAs is observed when increasing the sodium concentration. The largest fall in Tm (approximately 15 degrees C) is observed for the tertiary transition of the initiator tRNA. Among various cations tested the following efficiency in the overall stabilization of tRNAPhe is observed: spermine greater than spermidine greater than putrescine greater than Na+ (approximately NH4+). Mg2+ is most efficient at concentrations above 5 mM, but below this concentration spermine and spermidine appear to be more efficient. The same hierarchy in stabilizing power of the polyamines and Na+ is observed for both transitions of the initiator tRNA. However, when compared with Mg2+, the polyamines are far less capable of stabilizing the tertiary structure. In contrast, spermine and spermidine are slightly better than Mg2+ in stabilizing the secondary structure. At increasing concentrations of the polyvalent cations (at fixed [Na+] ) the Tm values of the tRNAs attain a constant value.     

Internal motions of transfer RNA: a study of exchanging protons by magnetic resonance

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H2O samples in D2O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CG11 of yeast tRNAPhe at 17 degrees C, or 5 minutes for U8-A14 of yeast tRNA(Asp) at 20 degrees C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other, fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA(Phe) has a lifetime of 14 ms at 17 degrees C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether short-lived or long-lived. Buffer effects have also been observed in poly(rA).poly(rU), for which we derive a lifetime of 2.5 ms at 27 degrees C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.     

A novel conformational change of the anticodon region of tRNAPhe (yeast).

The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method. In the range between -15 degrees C and +30 degrees C A NOVEL CONFORMATIONAL TRANSITION OF THE TRNA could be characterized. This conformational change was found in the absence of any artificial label; it is a characteristic property of tRNAPhe in its native structure. This transition accounts for 30% of the total fluorescence change. Its activation enthalpy is 16 kcal/mole (67 kJ/mole), and the transition enthalpy is between -2 kcal/mole and +2 kcal/mole (+/-8 kJ/mole). A model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop. The experimental findings are discussed with respect to several hypotheses about the molecular mechanism of protein biosynthesis which postulate conformational rearrangements of the anticodon loop.     

Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsed FT-NMR double resonance studies of yeast tRNAPhe: specific nuclear Overhauser effects and reinterpretation of low temperature relaxation data.

Cross-relaxation effects are demonstrated between the imino protons and other protons in yeast tRNAPhe and H2O. A detailed examination has been made of the observed relaxation rate of the proton resonance at 11.8 ppm from DSS as a function of the D2O content in the solvent. This result, as well as the size and number of observed nuclear Overhauser effects, suggests that dipolar magnetization transfer between solvent H2O, amino, imino, and other tRNA protons may dominate the relaxation processes of the imino protons at low temperature. At higher temperatures the observed relaxation rate is dominated by chemical exchange. The selective nuclear Overhauser effects are shown to be an important aid in resonance assignments. By these means we were able to identify tow protons from the wobble base pair GU4 at 11.8 ppm and 10.4 ppm.     

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.     

Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom.

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 nuclease under near physiological conditions, and the resultant fragments are than electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe an E, coli tRNAfMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.     

1H NMR of valine tRNA modified bases. Evidence for multiple conformations.

Methyl and methylene protons of dihydrouridine 17 (hU), 6-methyladenosine 37 (M6A), 7-methylguanosine 46 (m7G), and ribothymidine 54 (rT) give clearly resolved peaks (220 MHz) for tRNA1val (coli solutions in D2O, 0.25 m NaCl, at 27 degrees C. Chemical shifts are generally consistent with a solution structure of tRNA1val similar to the crystal structure of tRNAphe (yeast). At least 3 separate transitions are observed as the temperature is raised. The earliest involves disruption of native tertiary structure and formation of intermediate structures in the m7G and rT regions. A second transition results in a change in structure of the anticodon loop, containing m6A. The final step involves unfolding of the m7G and rT intermediates and melting of the TpsiC helix. Low salt concentrations produce multiple, partially denatured conformations, rather than a unique form, for tRNA1val. Native structure is almost completely reformed by addition of Na+ but Mg2+ is required for correct conformation in the vicinity of m7G.