Shape Of tRNA Research Articles

TRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

TRNA Modifications: Impact on Structure and Thermal Adaptation.

Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation.

Multilevel functional and structural defects induced by two pathogenic mitochondrial tRNA mutations

Point mutations in hmtRNAs (human mitochondrial tRNAs) can cause various disorders, such as CPEO (chronic progressive external ophthalmoplegia) and MM (mitochondrial myopathy). Mitochondrial tRNALeu, especially the UUR codon isoacceptor, is recognized as a hot spot for pathogenic mtDNA point mutations. Thus far, 40 mutations have been reported in hmtRNAsLeu. In the present paper, we describe the wide range of effects of two substitutions found in the TΨC arms of two hmtRNAsLeu isoacceptors. The G52A substitution, corresponding to the pathogenic G12315A mutation in tRNALeu(CUN), and G3283A in tRNALeu(UUR) exhibited structural changes in the outer corner of the tRNA shape as shown by RNase probing. These mutations also induced reductions in aminoacylation, 3'-end processing and base modification processes. The main effects of the A57G substitution, corresponding to mutations A12320G in tRNALeu(CUN) and A3288G in tRNALeu(UUR), were observed on the aminoacylation activity and binding to hmEF-Tu (human mitochondrial elongation factor Tu). These observations suggest that the wide range of effects may amplify the deleterious impact on mitochondrial protein synthesis invivo. The findings also emphasize that an exact understanding of tRNA dysfunction is critical for the future development of therapies for mitochondrial diseases.

Subsites for substrate recognition by bacterial ribonuclease P

We have prepared series of shape variant RNAs of a tRNA precursor and analyzed the substrate shape recognition by bacterial ribonuclease P ribozyme and holoenzyme. The results showed the evidence for the presence of subsites for the recognition of the trna shape. We will discuss a new model for substrate recognition and the role of the protein component.

Model of the structure of the eukaryotic ribosomal translation termination complex

Models of the atomic structure of the eukaryotic translation termination complex containing mRNA, P-site tRNAPhe, human class 1 release factor eRF1, and 80S ribosome, were constructed by computational modeling. The modeling was based on the assumed structural-functional similarity between the tRNA and eFR1 molecules in the ribosomal A site. The known atomic structure of the 70S ribosome complexed with mRNA as well as the P-and A-site tRNAsPhe was used as a structural template for the modeling. The eRF1 molecule bound in the A site undergoes substantial conformational changes so that the mutual configuration of the N and M domains matches the overall tRNA shape. Two models of eRF1 binding to mRNA at the A site in the presence of P-site tRNAPhe were generated. A characteristic of these models is complementary interactions between the mRNA stop codon and the grooves at different sides of the surface of the eRF1 fragment, containing helix α2, NIKS loop, and helix α3 of the N domain. In model 1, the nucleotides of the mRNA stop codon at the A site are approximately equidistant (∼15 A) from the N (motifs NIKS and YxCxxxF) and C domains. In model 2, the stop codon is close to the N-domain motifs NIKS and YxCxxxF. Both models fit genetic and biochemical experimental data. The choice of a particular model requires additional studies.

Rapid ribosomal translocation depends on the conserved 18-55 base pair in P-site transfer RNA

The L shape of tRNA is stabilized by the 'tertiary core' region, which contains base-pairing interactions between the D and T loops. Distortions of the L shape accompany tRNA movement across the ribosomal surface. Here, using single-turnover rapid kinetics assays, we determine the effects of mutations within the tertiary core of P site-bound tRNA(fMet) on three measures of the rate of translocation, the part of the elongation cycle involving the most extensive tRNA movement. Mutations in the strictly conserved G18.U55 base pair result in as much as an 80-fold decrease in the rate of translocation, demonstrating the importance of the 18-55 interaction for rapid translocation. This implicates the core region as a locus for functionally important dynamic interactions with the ribosome and leads to the proposal that translocation of ribosome-bound tRNAs may be sequential rather than concerted.

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer–editing conformation

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.

Crystal structure of elongation factor P from Thermus thermophilus HB8.

Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-A resolution. EF-P consists of three beta-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N- and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an "L" shape, with 65- and 53-A-long arms at an angle of 95 degrees, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.

Escherichia coli tRNAs are resistant to the hyperprocessing reaction of homologous E. coli ribonuclease P ribozyme.

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the Km value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.

Is tRNA Binding or tRNA Mimicry Mandatory for Translation Factors?

tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

Molecular mimicry between protein and tRNA.

Mimicry is a sophisticated development in animals, fish, and plants that allows them to fool others by imitating a shape or color for diverse purposes, such as to prey, evade, lure, pollinate, or threaten. This is not restricted to the macro-world, but extends to the micro-world as molecular mimicry. Recent advances in structural and molecular biology uncovered a set of translation factors that resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic code. Nature must have evolved this art of molecular mimicry between protein and ribonucleic acid by using different protein structures until the translation factors sat in the cockpit of a ribosome machine, on behalf of tRNA, and achieved diverse actions. Structural, functional, and evolutionary aspects of molecular mimicry will be discussed.

Functional mapping of ribosome-contact sites in the ribosome recycling factor: a structural view from a tRNA mimic.

Ribosome recycling factor (RRF) is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of RRF resembles a tRNA shape, with an architecturally different flexibility compared with tRNA, but its structure-and-function relationships are unknown. We here found that an RRF variant defective in ribosome binding regains the binding capacity through 20 independent secondary changes occurring in three topologically distinct regions of RRF. Because two of these regions are equivalent to the tip of the anticodon stem and the upper surface of the acceptor stem of tRNA, RRF may interact with the ribosome in a way similar to tRNA, spanning 30S and 50S subunits, to exert its action for splitting the ribosome.

Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

Coordination of tRNA nuclear export with processing of tRNA.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease

To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

The 2.9 Å Crystal Structure of T. thermophilus Seryl-tRNA Synthetase Complexed with tRNA Ser

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

Conformations of t-RNA: base pairing and stacking.

The Phe t-RNA structure can be fit with one point per nucleotide to lattice models, and a fit for the 76 points to a face-centered cubic lattice is achieved with an RMS of 1.76 A. There are 32 chain folds possible upon these points. Because it is impossible to calculate directly all combinations of potential base pairs for these cases, an alternative is to determine low energy secondary structures and subsequently the tertiary pairs. For each lattice fold, the low energy secondary structures are generated from a list of proximal bases. From the lists of remaining possible tertiary pairs, all combinations are generated, and these include 2,365,440 allowed conformers. Among the possible types of non-native conformational variations observed is slip pairing, accompanied by a bulge, at the end of a stem. Small changes in secondary structure can result in different tertiary pairs. Other calculations, not constrained to the t-RNA shape, are presented that involve the packing of rigid stems on a flexible internal loop. For a simple cubic lattice there are 36,484,128 lattice folds for the sixteen bases enclosing the internal loop. By attaching rigid stems and accounting for their excluded volume these are reduced to only 258,979 possible configurations. The most common stacking arrangements involve the usual two pairs of stacked stems indicated in the crystal structure. The present enumerations suggest that a completely thorough exploration of three dimensional RNA structures is feasible only with prior specification of restrictions on conformational freedom, such as those given by secondary structures.

Intensity‐fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process

AbstractWe have measured the translational (DT) and rotational (DR) diffusion coefficients of bulk tRNA from baker's yeast during the thermal unfolding process by means of photon‐correlation spectroscopy. It should be noted that our estimate of the rotational diffusion coefficient represented, for the first time, measurements on a small macromolecule in solution by the photoelectron time‐of‐arrival technique with a delay‐time resolution of 1 nsec. The melting curves expressed in terms of δDT vs temperature were consistent with the literature data in revealing the melting steps and their dependence on NaCl concentration. Additionally, it was possible to prove the existence of an intermediate, more compact structure during the initial steps of the thermal unfolding process. We found that the temperature ranges over which this intermediate structure appears depend strongly on salt concentration. By utilizing both translational and rotational diffusion coefficients and Perrin's equations for ellipsoids of revolution, we have computed the values of the equivalent length and width of tRNA molecules in solution at four different temperatures for NaCl concentrations of 0.2, 0.5, and 1M. The approximate model of ellipsoids of revolution also permits us to obtain an estimate of the radius of gyration, which is in very good agreement with literature data measured by means of small‐angle x‐ray scattering. Furthermore, we have measured the shape and size changes of tRNA with varying NaCl concentrations at room temperatures (25°C). The molecule becomes smaller and more spherical when NaCl concentration increases. As a result of partial melting at 70°C, the macromolecule is surprisingly elongated with an approximate axial ratio of 8:1 and has dimensions of about 180/22Å. Such information on conformational changes by a simultaneous determination of rotational and translational diffusion coefficients illustrates the potential of this approach, not available by other methods.

Loss of methyl acceptor activity of ultraviolet-irradiated tRNA.

THE features responsible for the recognition of specific sites for methylation by tRNA methylases are presently not understood. Studies by Kuchino and Nishimura suggest that a specific octanucleotide sequence is recognised by rat liver guanosine methylases1. Furthermore, reports from Nishimura's laboratory show that a large (three-quarter) fragment from E. coli methionine tRNA could be methylated enzymatically2. Shershneva et al.3, however have shown that fragments are not methylated until recombined. Studies in our laboratory have demonstrated that certain fragments from a mixture of E. coli4 tRNAs can serve as substrate for rat liver tRNA methylases. These fragments are believed to have an average S value of 0.64 and therefore would be about ten nucleotides long. Using ultraviolet-induced alterations, we have investigated further the importance of the molecular shape of tRNA as it affects its recognition by tRNA methylases.