Study on the conformational state of Escherichia coli tRNA-Phe in solution by ESR-spectometry without modulation

ESR-spectrometry without modulation of the magnetic field was used for registering the EST spectral line shape (with shape distortion about 0.1 percent) of spin-labeled Escherichia coli tRNAPhe. The analysis of line shape of two different spin-labels in position 8 (S4U) revealed that tRNAPhe in solution always exists as a mixture of at least two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and the biological state of tRNA (deacylated, aminoacyl- or peptidyl-tRNA). There are no large structural rearrangements upon aminoacylation or peptidylation of tRNA, the observed small changes of spectral line shape are due to the changes in conformational equilibria.     

Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities.

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.     

The interaction between biologically inactive tRNA conformers and leucyl-tRNA synthetase from rabbit liver

The interaction between tRNA conformers inactive in aminoacylation and leucyl-tRNA synthetase has been investigated. Heat inactivation of the enzyme in the presence of inactive tRNA conformers is shown to lead to a marked increase of inactivation rate while active tRNA conformers, on the other hand, reveal a protecting effect. To study the properties of the enzyme complexed with different tRNA conformers limited proteolysis has been used. Active tRNA conformers are found to protect leucyl-tRNA synthetase against hydrolysis while inactive ones tend to intensify it. Inactive tRNA conformers are also shown to inhibit the aminoacylation of native tRNA in vitro. On the basis of these data biologically inactive conformers of animal tRNA are assumed to form an unproductive complex with leucyl-tRNA synthetase and the structure of the enzyme involved in such interaction is supposed to be more labile and 'extended' than that in complex with active tRNA conformers.     

NMR line shapes and multi-state binding equilibria

Biological function of proteins relies on conformational transitions and binding of specific ligands. Protein-ligand interactions are thermodynamically and kinetically coupled to conformational changes in protein structures as conceptualized by the models of pre-existing equilibria and induced fit. NMR spectroscopy is particularly sensitive to complex ligand-binding modes-NMR line-shape analysis can provide for thermodynamic and kinetic constants of ligand-binding equilibria with the site-specific resolution. However, broad use of line shape analysis is hampered by complexity of NMR line shapes in multi-state systems. To facilitate interpretation of such spectral patterns, I computationally explored systems where isomerization or dimerization of a protein (receptor) molecule is coupled to binding of a ligand. Through an extensive analysis of multiple exchange regimes for a family of three-state models, I identified signature features to guide an NMR experimentalist in recognizing specific interaction mechanisms. Results show that distinct multi-state models may produce very similar spectral patterns. I also discussed aggregation of a receptor as a possible source of spurious three-state line shapes and provided specific suggestions for complementary experiments that can ensure reliable mechanistic insight.     

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.     

Solvent effect on the stability of mannobiose conformers

The conformational equilibria of seven methyl β-D -mannobioside conformers have been studied theoretically in five solvents. The structure of each individual conformer has been refined by the PCILO quantum-chemical method from the seven distinct low-energy regions determined from (Φ, Ψ) maps calculated by a potential function method. The stability of the conformers in dilute solution has been evaluated by using a method that consists of electrostatic, dispersion, and cavity terms. The calculated abundance of conformers depends on the solvent and results indicate that the preponderant conformer in the solution may not be the one adopted by mannobiose in the crystalline form. Based on the determined abundance of conformers, thermodynamically averaged nmr parameters, dipole moment, and linkage rotation have been calculated. The solvation behavior of methyl β-D -mannobioside is compared to those previously estimated for cellobiose and maltose.     

Paramagnetism in melanins: pH dependence

Changes in the paramagnetic properties of aqueous suspensions of melanin polymers have been monitored over a pH range from 1 to 12. Distinct changes in spin concentration and electron spin resonance spectral parameters (effective g value and line shape) are shown to occur. These data are interpreted in terms of pH- and temperaturedependent equilibria between diamagnetic and paramagnetic units on the melanin polymer, which can be partly or completely quenched if the melanin is precipitated by lowering the pH or by increasing the salt concentration. The heterogeneity of these units and possible chemical structures are discussed.     

Electrical potential of transfer RNAs: codon-anticodon recognition

Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength. The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA. The initiator tRNA is surrounded by uniformly spaced contours of negative potential. The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface. This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength. The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule. This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA. The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA. Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA.     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-α-D-idopyranose ring

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-α-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a 4C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity. In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1ρ measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-peneta-O-acetyl-α-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the 4C1 and 0S2 conformers. 13C NMR line shape analysis yields a ΔH+ of 40 kJ mol-1 and a ΔS‡ of 31 J mol-1 K-1 for the 4C1→0S2 interconversion and a ΔH‡ of 31 kJ mol-1 and a ΔS‡ of 13 J mol-1K-1 for the 0S2→4C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature. This revised version was published online in November 2006 with corrections to the Cover Date.     

Influence of various tRNA conformers on mRNA translation in cell-free protein-synthesizing systems

It is found that both globin mRNA translation in the wheat germ system and reticulocyte lysate protein synthesis are inhibited by the excess of exogenous tRNA. The inhibition was more expressed in the presence of inactive tRNA conformers. The data obtained confirm the possibility in principle of inactive tRNA conformers participation in negative regulation of protein synthesis.     

Feedback inhibition of the DAHP synthetases by tRNA in Saccharomyces cerevisiae

Summary The use of tyrosine inhibited and phenylalanine inhibited mutants in yeast makes possible the study of the properties of the two 3 deoxy-D-arabino-heptulosonic-acid-7-phosphate synthetases separately. The measurement of the activity of these two enzymes revealed the presence of endogenous inhibitors of the reaction in the crude extracts. These inhibitors are non dialysable, thermostable and sensitive to an RNAse treatment. The use of purified tRNAs showed that the phenylalanine sensitive enzyme is inhibited by both charged and uncharged phenylalanyl tRNA whereas the tyrosine sensitive enzyme is inhibited only by charged tyrosyl tRNA whereas the tyrosine sensitive enzyme is inhibited only by charged tyrosyl tRNA. Both enzymes are also inhibited by the free amino acids, i.e. respectively tyrosine and phenylalanine.     

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Theoretical studies on the conformation of saccharides. IV. Solvent effect on the stability of β-maltose conformers

The conformational equilibria of four β-maltose conformers have been studied theoretically in 12 solvents. The stability of the conformers in dilute solution has been compared by using the continuum reaction field method. The solvation energy consists of electrostatic, dispersion, and cavity terms, which have been determined from the calculated properties of β-maltose and physicochemical properties of solvents. The calculated population of four conformers significantly depends on the solvent (e.g., in dioxane, M1:M2:M3:M4 = 53.3: 20.3:17.7:8.7; in dimethyl sulfoxide, 40.1:21.8:22.8:15.3; and in water, 25.7:17.5:26.3:30.5) and was found to correlate with experimentally observed data. The results obtained indicate that the conformation adopted by β-maltose in the crystalline form is not the one preferred in solution. The roles of the individual contributions to the solvation energy have been analyzed. Based on the determined abundance of conformers, averaged residual optical activity and vicinal carbon–proton coupling constants have been calculated and discussed from the point of view of the solution behavior of β-maltose.     

Initiation of in vivo protein synthesis with non-methionine amino acids

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.     

The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.     

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)