Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe)

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.     

Characterization of the overall and internal dynamics of short oligonucleotides by depolarized dynamic light scattering and NMR relaxation measurements

The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The hydrodynamic diameter of the octamer and dodecamer is 20.5 +/- 1.0 A, assuming a rise per base of 3.4 A. The tridecamer, d(CGCGTTGTTCGCG), adopts a hairpin structure with nearly spherical dimensions and a diameter of 23.0 +/- 2.0 A. The DDLS relaxation measurements provide a powerful method for distinguishing between different conformations of the oligonucleotides (e.g., DNA double-helix versus hairpin structure). Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.     

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA. A 19F nuclear magnetic resonance relaxation study

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

On singularities of molecular relaxation in water solutions

In this paper, we analyzed spectra of liquid water and water solutions in a frequency domain, characteristic of the collective dynamics of water molecules (from 0 to 200 cm^?1). Particular attention is paid to the relaxation processes, one of which is observed in the terahertz region of the spectrum (～5–50 cm^?1). The physical essence of this process at the molecular level is still unclear. Based on data obtained this process is strongly suggested to interpret as a monomolecular relaxation of unbound water molecules.     

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.     

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.     

Role of Low-Molecular-Weight Substrates in Functional Binding of the tRNAPhe Acceptor End by Phenylalanyl-tRNA Synthetase

The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence. Human and E. coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end. Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E. coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case. The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide. The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm. The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.     

Dynamics of the RNA hairpin GNRA tetraloop

The dynamics of RNA hairpin tetraloops of the GNRA type [sequence G- any ribonucleotide (N)-purine (R)-A] was analyzed by fluorescence spectroscopy and by fluorescence-detected temperature-jump relaxation, using RNA oligomers with 2-aminopurine (2AP) substituted in two different positions of the loop sequence, Gp2APpApA (HP1) and GpAp2APpA (HP2), as indicator. The fluorescence of HP1 is much higher than that of HP2, indicating a lower degree of 2AP-stacking in HP1. Addition of Mg(2+) or Ca(2+) ions leads to an increase of fluorescence in HP1, whereas a decrease of fluorescence is observed in HP2. In both cases at least two ion-binding equilibria are required to fit titration data. T-jump experiments using fluorescence detection show a relaxation process with a time constant of 22 micros for HP1, whereas two relaxation processes with time constants 5 and 41 micros, are found for HP2. These results clearly demonstrate the existence of more than the single conformation state detected by NMR analysis. The T-jump amplitudes decrease with increasing bivalent ion concentration, indicating that one of the states is favored in the presence of bivalent ions. The loop relaxation processes are slower than standard stacking processes, probably because of activation barriers imposed by a restricted mobility of loop residues, and are assigned to a stacking rearrangement, probably between the 5' and the 3'-side. A similar process has been observed previously for the anticodon loop of tRNA(Phe). The rate constants of the transition are in the range of 10(4) s(-1) in the case of HP1. The data demonstrate the existence of structures that are not resolved by standard NMR because of fast exchange and are not found by X-ray analysis because of restrictions by crystal packing.     

A nature of conformational changes of yeast tRNA(Phe). High hydrostatic pressure effects

We analysed conformational changes of yeast tRNA(Phe) induced by high hydrostatic pressure (HHP) measured by Fourier-transform infrared (FTIR) and fluorescence spectroscopies. High pressure influences RNA conformation without other cofactors, such as metal ions and salts. FTIR spectra of yeast tRNA(Phe) recorded at high hydrostatic pressure up to 13 kbar with and without magnesium ions showed a shift of the bands towards higher frequencies. That blue shift is due to an increase a higher energy of bonds as a result of shortening of hydrogen bonds followed by dehydration of tRNA. The fluorescence spectra of Y-base tRNA(Phe) at high pressure up to 3 kbar showed a decrease of the intensity band at 430 nm as a consequence of conformational rearrangement of the anticodon loop leading to exposure of Y-base side chain to the solution. We suggest that structural transition of nucleic acids is driven by the changes of water structure from tetrahedral to a cubic-like geometry induced by high pressure and, in consequence, due to economy of hydration.     

Molecular dynamics of phenylalanine transfer RNA

The atomic motions of yeast phenylalanine transfer RNA have been simulated using the molecular dynamics algorithm. Two simulations were carried out for a period of 12 picoseconds, one with a normal Van der Waals potential and the other with a modified Van der Waals potential intended to mimic the effect of solvent. An analysis of large scale motions, surface exposure, root mean square displacements, helical oscillations and relaxation mechanisms reveals the maintenance of stability in the simulated structures and the general similarity of the various dynamic features of the two simulations. The regions of conformational flexibility and rigidity for tRNA(Phe) have been shown in a quantitative measure through this approach.