Anticodon Hairpin Research Articles

Glycyl-tRNA Synthetase as a Potential Universal Regulator of Translation Initiation at IRES-I

A full analysis has been conducted of the sequences and secondary structures of viral type-I or related IRESs identified in all of the elements that correspond to the previously described minimal fragment of the enterovirus C IRES, which mimics the glycine tRNA anticodon hairpin in the IRES structure and is necessary for the specific binding of glycyl-tRNA synthetase. Experiments on human glycyl-tRNA synthetase binding with the mRNA fragments of several taxonomically distant viruses showed that the binding constants of these complexes are similar. These results indicate that the regulation of translation initiation via glycyl-tRNA synthetase must be a universal mechanism for these viruses and the corresponding parts of their mRNAs must have similar spatial structures. Furthermore, at least one additional mRNA hairpin with the glycyl anticodon loop has been found in all analyzed viral type-I IRESs. It seems plausible that this extra hairpin is associated with the second RNA-binding site of the glycyl-tRNA synthetase dimer and stabilizes its complex with the viral mRNA.

An integrated, structure- and energy-based view of the genetic code.

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon–anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson–Crick pairs in the codon–anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon–anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.

TRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA(Ala)GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA(Ala)GGC sequences. This suggests that the sequences of bacterial tRNA(Ala)GGC have evolved to avoid reading incorrect codons.

Prediction of folding nuclei in tRNA molecules

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.

Particle-Based Implicit Solvent Model for Biosimulations: Application to Proteins and Nucleic Acids Hydration.

In addition to the simulation of two proteins described previously, we report on the application of our recently developed particle-based implicit solvent model to the simulations of four nucleic acid molecules, the 17 bases anticodon hairpin of the Asp-tRNA, the decamer d(CCGCCGGCGG) in both A and B form, and the containing EcoRI restriction site dodecamer d(CGCGAATTCGCG). The solvent is represented by a fluid of Lennard-Jones polarizable pseudoparticles of molecular size, the induced dipoles of which are sensitive to the solute electric field but not to each other. When implemented in a molecular dynamics algorithm with the Amber94 force field, the model allows to simulate efficiently the conformational evolution of the nucleic acids, yielding stable three-dimensional structures in agreement with experiments and other simulations in explicit solvent. In the same run, it is also able to provide estimations of the electrostatic solvation free energy within short time windows which correlate well with the Poisson-Boltzmann calculations. In addition, the molecular aspect of the solvent model allows for the reproduction of the highly localized water molecules in the major or minor grooves of the nucleic acid double helices, despite the absence of explicit water hydrogen bonds.

Sequence-specific RNA cleavage by oligonucleotide-peptide conjugates

This paper considers the site-directed cleavage of the in vitro transcript of human tRNALys3 by the conjugate CNH2Gly(ArgLeu)3ArgLeuN—p-DAG—5"CCCTGGACCCTCAGAT3" (conjugate pep-1A) of the oligodeoxyribonucleotide 1A (CCCTGGACCCTCAGAT) with a peptide [LeuArg]4Gly attached at the 5" terminus of the oligonucleotide through diethylene glycol as a linker. Under physiological conditions, the conjugate pep-1A cleaved the C56-A57 phosphodiester bond of the RNA target with high efficacy. In addition, the conjugate cleaved several other bonds in the D loop, at the basis of the amino-acceptor stem, and in the anticodon hairpin due, probably, to the formation of imperfect complexes of the conjugate with partially complementary sequences in the RNA structure.

Why Termination tRNAs Have Not Been Found? Because They Are Hidden in the Large Ribosomal RNA (A Hypothesis)

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.

RNA hydration: three nanoseconds of multiple molecular dynamics simulations of the solvated tRNAAsp anticodon hairpin

The hydration of the tRNAAsp anticodon hairpin was investigated through the analysis of six 500 ps multiple molecular dynamics (MMD) trajectories generated by using the particle mesh Ewald method for the treatment of the long-range electrostatic interactions. Although similar in their dynamical characteristics, these six trajectories display different local hydration patterns reflecting the landscape of the “theoretical” conformational space being explored. The statistical view gained through the MMD strategy allowed us to characterize the hydration patterns around important RNA structural motifs such as a G-U base-pair, the anticodon U-turn, and two modified bases: pseudouridine and 1-methylguanine. The binding of ammonium counterions to the hairpin has also been investigated. No long-lived hydrogen bond between water and a 2′-hydroxyl has been observed. Water molecules with long-residence times are found bridging adjacent pro-Rp phosphate atoms. The conformation of the pseudouridine is stiffened by a water-mediated base-backbone interaction and the 1-methylguanine is additionally stabilized by long-lived hydration patterns. Such long-lived hydration patterns are essential to ensure the structural integrity of this hairpin motif. Consequently, our simulations confirm the conclusion reached from an analysis of X-ray crystal structures according to which water molecules form an integral part of nucleic acid structure. The fact that the same conclusion is reached from a static and a dynamic point of view suggests that RNA and water together constitute the biologically relevant functional entity.

H-bond stability in the tRNA(Asp) anticodon hairpin: 3 ns of multiple molecular dynamics simulations

Multiple molecular dynamics trajectories of the solvated and neutralized 17-residue tRNA(Asp) anticodon hairpin were generated for a total of 3 ns. Explicit treatment of all long-ranged electrostatic interactions by the particle mesh Ewald algorithm, as implemented in the AMBER MD software package, effected a degree of structural stabilization not previously achieved by use of a long 16-A solvent interaction truncation scheme. The increased stability of this multiple molecular dynamics set was appropriate for an in-depth analysis of the six 500-ps-long trajectories and allowed the characterization of a number of key structural interactions. The dynamical behavior of the standard Watson-Crick base pairs, the noncanonical G30-U40 "wobble" base pair, and the psi 32-C38 pseudo-base pair is presented as well as that of two C--H... O hydrogen bonds found to contribute to the array of tertiary interactions that stabilize the seven-nucleotide native loop conformation. The least mobile residue in the loop is U33, which forms the U-turn motif and which participates in several hydrogen-bonding interactions, whereas the most mobile residue is the apical residue G34 at the wobble position, a factor undoubtedly important in its biological function. The set of multiple molecular dynamics trajectories obtained does not converge on a 500-ps time scale to a unique dynamical model but instead describes an ensemble of structural microstates accessible to the system under the present simulation protocol, which is the result of local structural heterogeneity rather than of global conformational changes.

Hydration of C-H groups in tRNA.

Molecular dynamic (MD) simulations of the anticodon hairpin of tRNA(Asp) and of the full tRNA, both in a solvent bath with neutralizing NH4+ counter-ions, have been produced with the particle mesh ewald (PME) method and the multiple molecular dynamics (MMD) strategy. The latter consists of generating uncorrelated trajectories starting from the same initial configuration but with a slightly perturbed initial velocity distribution. The 3 ns (six uncorrelated 500 ps MD trajectories) MMD set of the 17 nucleotide anticodon fragment and the single 500 ps trajectory of the 75 nucleotide tRNA were analyzed with the aim of characterizing long lived C-H...Ow interactions for the two main nucleic acid base and ribose C-H bond types. Some C-H sites present very long residence lifetimes for water molecules, especially those around the ribose H(3') and the pyrimidine H(5) atoms. The C(3')-H(3')...Ow contacts occur concurrently with the strong hydration of the anionic phosphate oxygen atoms and especially with the water bridges linking successive phosphate groups along the polynucleotide chain. Therefore, these contacts are of opportunistic character and result from the geometries of the covalent structure and adjacent interactions. On the other hand, the pyrimidine H(5) atoms display a hydrophilic character with interaction geometries indicating that water contacts in which they are involved should be considered as bona fide hydrogen bonds.

Modified nucleoside-dependent transition metal binding to DNA analogs of the tRNA anticodon stem/loop domain.

Biologically active DNA analogs of tRNAPhe (tDNAPhe) were used to investigate metal ion interaction with tRNA-like structures lacking the 2'OH. Binding of Mg2+ to the 76 oligonucleotide tDNAPhe, monitored by circular dichroism spectroscopy, increased base stacking and thus the conformational stability of the molecule. Mg2+ binding was dependent on a d(m5C) in the anticodon region. In contrast to Mg2+, Cd2+ decreased base stacking interactions, thereby destabilizing the molecule. Since alterations in the anticodon region contributed to most of the spectral changes observed, detailed studies were conducted with anticodon hairpin heptadecamers (tDNAPheAC). The conformation of tDNAPheAC-d(m5C) in the presence of 1 mM Cd2+, Co2+, Cr2+, Cu2+, Ni2+, Pb2+, VO2+ or Zn2+ differed significantly from that of the biologically active structure resulting from interaction with Mg2+, Mn2+ or Ca2+. Nanomolar concentrations of the transition metals were sufficient to denature the tDNAPheAC-d(m5C) structure without catalyzing cleavage of the oligonucleotide. In the absence of Mg2+ and at [Cd2+] to [tDNAPheAC-d(m5C)] ratios of approximately 0.2-1.0, tDNAPheAC-d(m5C40) formed a stable conformation with one Cd2+ bound with a Kd = 3.7 x 10(-7) M. In contrast to Mg2+, Cd2+ altered the DNA analogs without discriminating between modified and unmodified tDNAPheAC. This ability of transition metals to disrupt higher order DNA structures, and possibly RNA, at microM concentrations, in vitro, demonstrates that these structures are potential targets in chronic metal exposure, in vivo.

Cloning of the 3'-terminal region encoding movement and coat proteins of a Korean isolate of odontoglossum ringspot virus.

The 3'-terminal 1855 nucleotides (nts) of a Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) were cloned and sequenced. The sequence contained two open reading frames, which encode the cell-to-cell movement protein (MP) and coat protein genes, and are 912 nts and 477 nts long, respectively. The MP gene contained a conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA locating between 1117 nts and 1292 nts from the 3'-end. The 3' untranslated region (UTR) of the virus comprises 414 nts, includes nine pseudoknots and a tRNA-like structure domain containing aminoacyl acceptor arm and the anticodon hairpin loop coding for histidine.

Defining a Smaller RNA Substrate for Elongation Factor Tu

A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA were obtained that bound T. thermophilus EF-Tu as well as Ala-tRNA(Ala). Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.

Replacement of RNA hairpins by in vitro selected tetranucleotides.

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA.

Structural domains of transfer RNA molecules.

In this article, we have described various detailed features of the conformation of yeast tRNA(Phe) revealed by recent refinement analysis of x-ray diffraction data at 2.5 A resolution. The gross features of the molecule observed in the unrefined version have been largely confirmed and a number of new features found. The unique role of the ribose 2' hydroxyl groups in maintaining a series of nonhelical conformations in this RNA molecule has become apparent. Many of these features are a direct consequence of the geometry of the ribose phosphate backbone of RNA molecules, and these may also be found in structured regions of other RNA species as well. Special attention has been directed toward two conformational motifs revealed by this analysis. These include the striking similarity between the TpsiC and anticodon hairpin turns in the polynucleotide chain, which are stabilized by the participation of uridine in the U turn. In addition, there is frequent occurrence of an arch conformation in the polynucleotide chian which is stabilized by hydrogen bonds from 2' hydroxyl residues to phosphate groups across the base of the arch. The importance of the 2' hydroxyl interactions in defining tertiary structure is illustrated by the fact that, in the nonhelical regions, almost half of the ribose residues are involved in O2' hydrogen-bonding interactions which stabilize the conformation of the molecule.

Investigation of the base‐pairing structure of the anticodon hairpin from E. coli initiator tRNA by high‐resolution nmr

AbstractThe high‐resolution nmr spectrum of the anticodon hairpin from E. coli tRNAfMet has been obtained at a number of different temperatures. The positions of the resonances from interior Watson‐Crick base pairs are well accounted for (within 0.1 ppm) by a semi‐empirical ring current shift theory, but the terminal base pairs are susceptible to the exact orientation of adjacent bases in single‐stranded regions. From a careful examination of the exact way in which resonances disappear at elevated temperatures, we conclude that melting in the nmr experiments occurs when the lifetime of a base pair is reduced to several milliseconds. On the basis of these experiments we are able to assign an nmr Tm to each individual base pair and these should be useful in interpreting the melting behavior of the intact molecule. An “extra” resonance is observed at ∼11.3 ppm and, on the basis of its position and temperature sensitivity, it is tentatively assigned to the ring nitrogen proton of a “protected” U residue in the anticodon loop. A strong preference for stacking of a nonbase‐paired A residue on an adjacent GC base pair is observed even at temperatures in excess of 52°C.