tRNA In Solution Research Articles

Atomic structure of the regulatory TGS domain of Rel protein from Mycobacterium tuberculosis and its interaction with deacylated tRNA.

The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel β-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery.

Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase.

The 2H phosphoesterase family contains enzymes with two His-X-Ser/Thr motifs in the active site. 2H enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. For many 2H enzymes, the physiological function is unknown. Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2′-AMP, 3′-AMP, phosphate, and NADP+. Comparisons to the well-characterized vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) highlight specific features of the catalytic cycle and substrate recognition in both enzymes. The role played by the helix α7, unique to CNPases within the 2H family, is apparently taken over by Arg130 in the bacterial enzyme. Other residues and loops lining the active site groove are likely to be important for RNA substrate binding. We visualized conformational changes related to ligand binding, as well as the position of the nucleophilic water molecule. We also present a low-resolution model of E. coli LigT bound to tRNA in solution, and provide a model for RNA binding by LigT, involving flexible loops lining the active site cavity. Taken together, our results both aid in understanding the common features of 2H family enzymes and help highlight the distinct features in the 2H family members, which must result in different reaction mechanisms. Unique aspects in different 2H family members can be observed in ligand recognition and binding, and in the coordination of the nucleophilic water molecule and the reactive phosphate moiety.

Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus.

The 3(') untranslated region (3(') UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined three-dimensional structure of a functional element in the 3(') UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.

Solution structure of DNA: The method of nuclear magnetic resonance spectroscopy

A method of NMR spectroscopy which involves the computation of magnetic shielding constants from xyz coordinates, taking into consideration the contribution to shielding from ring current fields, the diamagnetic and paramagnetic components of the atomic magnetic anisotropy, is presented to solve the solution spatial configuration of single stranded and double helical nucleic acids. It is shown that the CCA terminus of tRNA in solution assumes a structure very much different from its crystal structure. However, for the double helical poly(dG-dC)-poly(dG-dC) in high salt solution, the observed structure shows eminent agreement with the left-handed Z-DNA proposed from single crystal structural studies.

In Silico Examination Of The Influence Of Nucleotide Modifications And Magnesium Ions On tRNA Structure And Dynamics

In our work the influence of chemical modificatons and ions on RNA structure and dynamics has been tested. The effect of nucleotide modifications on E. coli and yeast tRNA in solution has been examined with a molecular dynamics approach. Simulations show a decrease of helical content in RNA secondary structure due to those modifications. In another step magnesium ion binding effects on the same tRNA were looked at by performing simulations with and without ions bound to tRNA. Ions coordinating nucleotides in those simulations show them highly affecting local secondary structure motifs. Thus the simulations performed give new hints on the function of nulceotide modifications and ion binding to RNA.

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding.

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.

Characterization of the tRNA and ribosome‐dependent pppGpp‐synthesis by recombinant stringent factor from Escherichia coli

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

Molecular Dynamics Simulations of Solvated Yeast tRNA Asp

Transfer RNA molecules are involved in a variety of biological processes, implying complex recognition events with proteins and other RNAs. From a structural point of view, tRNAs constitute a reference system for studying RNA folding and architecture. A deeper understanding of their structural and functional properties will derive from our ability to model accurately their dynamical behavior. We present the first dynamical model of a fully neutralized and solvated tRNA molecule over a 500-ps time scale. Starting from the crystallographic structure of yeast tRNA Asp, the 75-nucleotide molecule was modeled with 8055 water molecules and 74 NH 4 + counterions, using the AMBER4.1 program and the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions. The calculations led to a dynamically stable model of the tRNA molecule. During the simulation, all secondary and tertiary base pairs are maintained while a certain lability of base triples in the tRNA core is observed. This lability was interpreted as resulting from intrinsic factors associated with the “weaker” hydrogen bonding patterns seen in these base triples and from an altered ionic environment of the tRNA molecule. Calculated thermal factors are used to compare the dynamics of the tRNA in solution and in the crystal. The present molecular dynamics simulation of a complex and highly charged nucleic acid molecule attests to the fact that simulation methods are now able to investigate not only the dynamics of proteins, but also that of large RNA molecules. Thus they also provide a basis for further investigations on the structural and functional effects of chemical and posttranscriptionally modified nucleotides as well as on ionic environmental effects.

Determination of the angle between the anticodon and aminoacyl acceptor stems of yeast phenylalanyl tRNA in solution.

A principal feature of the crystal structures of tRNAs is an L-shaped tertiary conformation in which the aminoacyl acceptor stem and the anticodon stem are approximately perpendicular. However, the anticodon-acceptor interstem angle has not been precisely quantified in solution for any tRNA. Such a determination would represent an important test of the predicted global conformation of tRNAs in solution. To this end, we have constructed a yeast tRNA(Phe) heteroduplex RNA molecule in which the anticodon and acceptor stems of the tRNA have each been extended by approximately 70 base pairs. A comparison of the rotational decay times of the heteroduplex molecule and a linear control yields an interstem angle of 89 +/- 4 degrees in 4 mM magnesium chloride/100 microM spermine hydrochloride, essentially identical to the corresponding angle observed in the crystal under similar buffer and temperature conditions. The current approach is applicable to the study of a wide variety of RNA molecules that possess elements of nonhelical structure.

Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation.

Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution.

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)

On the coordination properties of Eu3+ bound to tRNA.

The luminescence properties of Eu3+ have been used to investigate the binding and coordination properties of the ion with tRNA, as an attempt to resolve the discussion of whether metal ions bind to tRNA in solution only by Debye-Hückel screening, or whether direct coordination to specific sites may occur. Binding studies with Escherichia coli tRNAmet/f (taking advantage of 4-thiouracil-sensitized Eu3+ emission) distinguish three classes of binding affinities. Two of these are single sites with affinities approx. 10(4) and approx. 10(3) tighter than the nonspecific affinity of Eu3+ for native DNA. Mg2+ competes for binding at both these sites. Measurement of the lifetime and excitation spectrum of Eu3+ bound to the highest affinity site shows that the ion has two to five non-phosphate ligands in its inner coordination sphere. The existence of this coordinated site demonstrates that electrostatic screening is not the only mechanism for metal ion interaction with tRNA. The coordination properties of the high-affinity Eu3+ site do not agree with the properties of any of the metal ion sites found in the two tRNAphe crystal forms. Possible reasons for this discrepancy are discussed; it may be that ions bind differently to isolated molecules in solution than to molecules packed in a crystal lattice.

The modelling of the decoding site of the Escherichia coli ribosome.

Individual ribosomal proteins S4, S9 and S13 were tested for their ability to interact with tRNA and synthetic polynucleotides. All three proteins bind to immobilized to Sepharose poly(A) and poly(U), while S4 and S13 form stoichiometric (1:1) complexes with tRNA in solution. We show that only the polynucleotide X S13 complexes are able to select their cognate tRNAs. In particular, the affinity of tRNAPhe to the binary poly(U) X S13 complex is about three orders of magnitude higher than that for poly(U) alone.

Chemical probes for tRNA tertiary structure: Comparative alkylation of tRNA with methylnitrosourea, ethylnitrosourea and dimethylsulfate

The tertiary structure of tRNA in solution can be proved by chemical modification experiments. Three reagents, N-ethyl- N-nitrosourea, N-methyl- N-nitrosourea and dimethylsulfate which are known to alkylate nucleic acids at nucleophilic centers were compared. It is found that N-ethyl- N-nitrosourea and N-methyl- N-nitrosourea mainly react with phosphate residues and dimethylsulfate only with the bases. With dimethylsulfate the extent of alkylation of guanosines is about one order of magnitude higher than that of the phosphates by the nitrosocompounds.

Study on conformational states of Escherichia coli tRNAPhe in solution by a modulation-free ESR-spectrometer.

A modulation free Electron Spin Resonance spectrometer was used for the registration of spectral absorption lines of a spin-labeled Escherichia coli phenylalanine tRNA in solution with low (less than 0.1%) line shape distortion. The analysis of line shape of two different spin-labels introduced into position 8 revealed that phenylalanine tRNA in solution exists as a mixture of two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and functional state of tRNA (deacylated, aminoacylated or peptidylated). There are no overall structural rearrangements upon aminoacylation or peptidylation of tRNA. The observed small changes of spectral line shape can be assigned to shifts in conformational equilibria.

Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt-free tRNA in solution.

AbstractThe influence of sodium ion concentration in solution on the initially salt‐free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (DT) and rotational (DR) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H2O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl2 solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low‐salt or salt‐free tRNA solutions, we detected in the linewidth distribution function an extra‐fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light‐scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long‐range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt‐free or low‐salt solutions.

High Resolution NMR Studies of the Structure and Dynamics of tRNA in Solution

Ribonucleic acids are composed of a sugar-phosphate backbone with bases attached at position l' of the sugar ring (Fig. 1). The backbone consists of six rotatable bonds defined by the angles 0/, cp', 1/1', 1/1, cp, and w, while the base and sugar rings are joined by the rotatable glycosidic bond, x. The four major bases are the purines guanine· (G) and adenine (A), and the pyrimidines cytosine (C) and uracil (U). Polynucleotide strands can interact with each other to form duplex structures by formation of complementary Watson-Crick adenine-uracil and guanine· cytosine base pairs. tRNA is composed of a single ribonucleic acid chain that ranges between 73 and 93 nucleotides in length with a terminal phosphate at its 5' end (residue 1). The nucleotide sequence of known tRNAs (2, 3) suggests that the polynucleotide chain can fold back on itself to generate double-stranded helical regions, as well as single­ stranded loop regions of the hairpin (chain reversal) and bulge types (Fig. 2)_ There are three hairpin loops defined as the D loop (contains the dihydrouracil base), the anticodon loop (contains the trinucleotide sequence complementary to the trinucleotide codon on messenger RNA), and the TljlC loop (contains the common sequence ribothymine-pseudouridine-cytosine). Each of these three loops is asso­ ciated with its corresponding double helical region called the stem. There is in addition a variable loop whose length depends on individual tRNAs and the acceptor stem whose 3' terminus is the site of aminoacylation. A survey of the known tRNA sequences demonstrates the presence of invariant and semi-invariant bases and they are defined in Figure 2 with purines and pyrimidines designated by Y and R, respectively (4, 5).

Dependence of tRNA structure in solution upon ionic condition of the solvent. Fluorescence studies of Mg2+ binding to tRNAPhe from barley embryos.

Barley phenylalanine tRNA (tRNAphe) exhibits a 4.7‐fold enhancement of fluorescence intensity in response to the addition of MgCl2 in 0.01 M Tris‐HCl, pH 7.5, 0.001 M Na2 EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaCl‐free tRNAphe solution strong interacting (Ks= 1.5 × 105 M−1n= 2.4) and weak independent (Kw= 9 x 103 M−1) Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. Ks and Kw, in the presence of NaCl, had lower values than in NaCl‐free solution, since Na+ was found to be a competitive inhibitor of Mg2+ binding. Fluorescence titrations of tRNAphe solutions containing Mg2+ evidenced 2 Na+ binding sites/Mg2+ binding site (at least in the region of weak sites of Mg2+) characterized by inhibitory binding constants K̄i1 Na= 45 M−1 and K̄i 1 Na= 8.5 M−1 (mean values) for first and second Na+ bindings respectively. Na+ exerts a destabilizing effect on tRNA conformation induced by Mg2+. A model of Mg2+ binding and concomitant conformational changes of the tRNA molecule is discussed: one class of Mg2+ binding sites in unfolded tRNA and two classes of sites, strong and weak, in folded conformation are postulated.

Properties of tRNA-Specific Antibodies from NZB/NZW Mice

Antibodies that bind tRNA are produced spontaneously in New Zealand Black/New Zealand White (NZB/NZW) F1 hybrid female mice. An assay for the detection of these antibodies has been developed by using gel filtration and radioactive tRNA. This assay was found superior to the widely used ammonium sulfate precipitation assay because of the nature of the interaction between the protein and the tRNA. The ant-bodies bound native tRNA preferentially to tRNA denatured by cross-linking with formaldehyde. This conformational specificity was confirmed in competition experiments. The antibodies to native tRNA had an average association constant of 5 x 10(7) leter/mole at 4 degrees C and could bind to more than one site per tRNA molecule. Experiments with immunoglobulin class-specific anti-mouse antisera, in solution and by radioimmunoelectrophoresis, showed that the antibodies were heterogeneous, but were predominantly of the IgG class. These antibodies may be useful for detection, localization, and conformational analysis of tRNA in solution as well as for understanding the pathogenesis of the lupus-like syndrome in these mice.

An NMR approach to tRNA tertiary structure in solution

Atomic coordinates of E. Coli tRNA1Val have been generated from the X-ray crystal structure of Yeast tRNAPhe by base substitution followed by idealization...