tRNA Core Research Articles

The modification landscape of Pseudomonas aeruginosa tRNAs.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional chemical modifications. Approximately 100 different modifications have been identified in tRNAs, and each tRNA typically contains 5-15 modifications that are incorporated at specific sites along the tRNA sequence. These modifications may be classified into two groups according to their position in the three-dimensional tRNA structure, i.e., modifications in the tRNA core and modifications in the anticodon-loop (ACL) region. Since many modified nucleotides in the tRNA core are involved in the formation of tertiary interactions implicated in tRNA folding, these modifications are key to tRNA stability and resistance to RNA decay pathways. In comparison to the extensively studied ACL modifications, tRNA core modifications have generally received less attention, although they have been shown to play important roles beyond tRNA stability. Here, we review and place in perspective selected data on tRNA core modifications. We present their impact on tRNA structure and stability and report how these changes manifest themselves at the functional level in translation, fitness and stress adaptation.

TRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Posttranscriptional modification to the core of tRNAs modulates translational misreading errors.

Protein synthesis on the ribosome involves successive rapid recruitment of cognate aminoacyl-tRNAs and rejection of the much more numerous incorrect near- or non-cognates. The principal feature of translation elongation is that at every step, many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translational accuracy require that cognate tRNAs have relatively similar acceptance rates by the ribosome. To achieve that, tRNAs evolved to compensate for differences in amino acid properties and codon-anticodon strength that affect acceptance. Part of that response involved tRNA posttranscriptional modifications, which can affect tRNA decoding efficiency, accuracy, and structural stability. The most intensively modified regions of the tRNA are the anticodon loop and structural core of the tRNA. Anticodon loop modifications directly affect codon-anticodon pairing and therefore modulate accuracy. Core modifications have been thought to ensure consistent decoding rates principally by stabilizing tRNA structure to avoid degradation; however, degradation due to instability appears to only be a significant issue above normal growth temperatures. We suspected that the greater role of modification at normal temperatures might be to tune tRNAs to maintain consistent intrinsic rates of acceptance and peptide transfer and that hypomodification by altering these rates might degrade the process of discrimination, leading to increased translational errors. Here, we present evidence that most tRNA core modifications do modulate the frequency of misreading errors, suggesting that the need to maintain accuracy explains their deep evolutionary conservation.

Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance

Post-transcriptional modifications have critical roles in tRNA stability and function1–4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2′-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2′-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

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.

TRNA evolution from the proto-tRNA minihelix world

ABSTRACTMultiple models have been advanced for the evolution of cloverleaf tRNA. Here, the conserved archaeal tRNA core (75-nt) is posited to have evolved from ligation of three proto-tRNA minihelices (31-nt) and two-symmetrical 9-nt deletions within joined acceptor stems (93 – 18 = 75-nt). The primary evidence for this conclusion is that the 5-nt stem 7-nt anticodon loop and the 5-nt stem 7-nt T loop are structurally homologous and related by coding sequence. We posit that the D loop was generated from a third minihelix (31-nt) in which the stem and loop became rearranged after 9-nt acceptor stem deletions and cloverleaf folding. The most 3´-5-nt segment of the D loop and the 5-nt V loop are apparent remnants of the joined acceptor stems (14 – 9 = 5-nt). Before refolding in the tRNA cloverleaf, we posit that the 3′-5-nt segment of the D loop and the 5-nt V loop were paired, and, in the tRNA cloverleaf, frequent pairing of positions 29 (D loop) and 47 (V loop) remains (numbered on a 75-nt tRNA cloverleaf core). Amazingly, after >3.5 billion years of evolutionary pressure on the tRNA cloverleaf structure, a model can be constructed that convincingly describes the genesis of 75/75-nt conserved archaeal tRNA core positions. Judging from the tRNA structure, cloverleaf tRNA appears to represent at least a second-generation scheme (and possibly a third-generation scheme) that replaced a robust 31-nt minihelix protein-coding system, evidence for which is preserved in the cloverleaf structure. Understanding tRNA evolution provides insights into ribosome and rRNA evolution.

TRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World.

Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA.

RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

Interrupted catalysis: the EF4 (LepA) effect on back translocation

EF4 (LepA) promotes back translocation of tRNAs on the ribosome, and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4‐catalyzed back translocation. EF4‐dependent back translocation proceeds from post‐translocation complex (POST) to pre‐translocation complex (PRE) via a four‐step kinetic scheme, i.e., POST → I1 → I2 → I3 → PRE, that is not the simple reverse of translocation, and during which movement of the 3’‐end of peptidyl–tRNA is partially decoupled from movements of the tRNA core regions and of mRNA. EF4 may be thought of as performing an interrupted catalysis of this process, stopping at the formation of I3 rather than catalyzing the complete process of back translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis. Supported by NIH grant GM071014 to B. S. C.

Exit Strategies for Charged tRNA from GluRS

For several class I aminoacyl-tRNA synthetases (aaRSs), the rate-determining step in aminoacylation is the dissociation of charged tRNA from the enzyme. In this study, the following factors affecting the release of the charged tRNA from aaRSs are computationally explored: the protonation states of amino acids and substrates present in the active site, and the presence and the absence of AMP and elongation factor Tu.Through molecular modeling, internal pKa calculations, and molecular dynamics simulations, distinct, mechanistically relevant post-transfer states with charged tRNA bound to glutamyl-tRNA synthetase from Thermus thermophilus (Glu-tRNAGlu) are considered. The behavior of these nonequilibrium states is characterized as a function of time using dynamical network analysis, local energetics, and changes in free energies to estimate transitions that occur during the release of the tRNA. The hundreds of nanoseconds of simulation time reveal system characteristics that are consistent with recent experimental studies.Energetic and network results support the previously proposed mechanism in which the transfer of amino acid to tRNA is accompanied by the protonation of AMP to H-AMP. Subsequent migration of proton to water reduces the stability of the complex and loosens the interface both in the presence and in the absence of AMP. The subsequent undocking of AMP or tRNA then proceeds along thermodynamically competitive pathways. Release of the tRNA acceptor stem is further accelerated by the deprotonation of the α-ammonium group on the charging amino acid. The proposed general base is Glu41, a residue binding the α-ammonium group that is conserved in both structure and sequence across nearly all class I aaRSs. This universal handle is predicted through pKa calculations to be part of a proton relay system for destabilizing the bound charging amino acid following aminoacylation. Addition of elongation factor Tu to the aaRS·tRNA complex stimulates the dissociation of the tRNA core and the tRNA acceptor stem.

Interrupted Catalysis: The EF4 (LepA) Effect on Back-Translocation

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST→I1→I2→I3→PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3′-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I3 rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis.

Identification of Cellular Pathways Mediating Progression of Monoclonal Gammopathy (MGUS) to Multiple Myeloma (MM) Using Gene Expression Profiling (GEP).

Events mediating transformation from pre-malignant MGUS to MM is currently not well defined. Recurrent genetic abnormalities such as t(11;14), t(4;14), hyperdiploidy and chromosome 13 deletion are already present in MGUS at relatively similar frequency to MM. The unified deregulation of D-type cyclins is also already present in MGUS. Previous GEP studies have revealed little differences between MGUS and MM. A more recent study using a large patient cohort and new generation Affymetrix genechip identifies an MGUS signature but the functional and biological significance underlying this signature is unknown. In the current study, we analyze a cohort of 22 MGUS and 101 MM from the Mayo Clinic with GEP performed on Affymetrix U133A genechip using gene set enrichment analysis, a method that analyze differentially expressed gene between 2 phenotypes of interest in the context of published or curated genesets that represent specific biological, chemical, or molecular perturbation to cells. This method increases the sensitivity of identifying low but significant changes in gene expression. We made further modification to the original method that allows assessment in individual samples rather than the average across a phenotype further increasing the specificity of the output. In this analysis, 313 genesets were significant enriched for genes over-expressed in MM compared to MGUS, representing potential activated pathways that mediate transformation. When MM samples and genesets were clustered using the enrichment score for each genesets and samples, 4 cluster of genesets emerged, one including a number of MYC genesets, one including a number of cell cycle related genesets, one including genesets related to metabolic activity and another including a number of IFN related genesets. Further dissection of these correlated genesets to identify common enriched genes (leading edge genes) led to identification of a MYC core signature, tRNA core signature, Proteosome core signature and metabolic core signature which are highly correlated. From known literature and biology, it is likely that MYC activation leads to downstream activation of protein synthesis (tRNA signature), degradation (proteasome signature), and metabolic pathway (metabolic signature). This is verified by GEP results from in vitro modulation of MYC activation in cell lines. In addition, a cell cycle core signature and IFN core signature was identified. Activation of IFN and MYC core signatures accounts for almost 90% of MM patients. The remaining patients have a metabolic signature without MYC or IFN activation. The activation of the different core signatures is significantly correlated with certain TC classes when assessed by Chi-Square test. IFN activation is significantly correlated with D1 subtypes and negatively correlated with D2 subtypes. MYC activation is negatively correlated with t(11;14). Similar patterns were observed in a validation dataset of 351 MM patients from UAMS (GSE2677) with GEP performed on the U133plus2.0 chip. These results are validated at the protein level using IHC on TMA of the Mayo cohort. The activation of the IFN and MYC pathway may represent predominant mechanism in MGUS to MM progression.

Structural basis for functional mimicry of long-variable-arm tRNA by transfer-messenger RNA

tmRNA and small protein B (SmpB) are essential trans-translation system components. In the present study, we determined the crystal structure of SmpB in complex with the entire tRNA domain of the tmRNA from Thermus thermophilus. Overall, the ribonucleoprotein complex (tRNP) mimics a long-variable-arm tRNA (class II tRNA) in the canonical L-shaped tertiary structure. The tmRNA terminus corresponds to the acceptor and T arms, or the upper part, of tRNA. On the other hand, the SmpB protein simulates the lower part, the anticodon and D stems, of tRNA. Intriguingly, several amino acid residues collaborate with tmRNA bases to reproduce the canonical tRNA core layers. The linker helix of tmRNA had been considered to correspond to the anticodon stem, but the complex structure unambiguously shows that it corresponds to the tRNA variable arm. The tmRNA linker helix, as well as the long variable arm of class II tRNA, may occupy the gap between the large and small ribosomal subunits. This suggested how the tRNA domain is connected to the mRNA domain entering the mRNA channel. A loop of SmpB in the tRNP is likely to participate in the interaction with alanyl-tRNA synthetase, which may be the mechanism for the promotion of tmRNA alanylation by the SmpB protein. Therefore, the tRNP may simulate a tRNA, both structurally and functionally, with respect to aminoacylation and ribosome entry.

Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA Pro, accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA Cys with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA Pro is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA Pro and tRNA Cys identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA Cys suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3′ end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.

Alternative design of a tRNA core for aminoacylation

The core of Escherichia coli tRNACys is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNAGln, which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNAGln, we sought to test and identify alternative functional design of the tRNACys core that contains G15:C48. Although analysis of the crystal structure of tRNACys and tRNAGln implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNACys with those in tRNAGln did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.

Probing a tRNA core that contributes to aminoacylation

The contribution of the tRNA “core” to aminoacylation is beginning to be recognized. One example is the core region of Escherichia coli tRNA Cys, which has been shown by biochemical studies to be important for aminoacylation. This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation factor EF-Tu and an analog of GTP. One of these layers consists of a 9:[13:22] base-triple, rather than the 46:[13:22] or 45:[13:22] base-triple that is commonly observed in tRNA structure. Because 13:22 is an important element in aminoacylation of E. coli tRNA Cys, a better understanding of its structure in the tRNA core will shed light on its role in aminoacylation. In this study, we used the phage T7 transcript of the tRNA as a substrate. We probed the structure of 13:22 by dimethyl sulfate and tested its partner in a base-triple by generating mutations that could be assayed for aminoacylation. The results of this study in general are in a better agreement with a 46:[13:22] base-triple that we previously proposed. Although these results are not interpreted as direct proof for the 46:[13:22] base-triple, they shed new light on features of the tRNA core that are important for aminoacylation.

Conservation of a tRNA core for aminoacylation.

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.

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.

Global flexibility of tertiary structure in RNA: yeast tRNAPhe as a model system.

The study of RNA structure using x-ray crystallography or NMR has yielded a wealth of detailed structural information; however, such approaches do not generally yield quantitative information regarding long-range flexibility in solution. To address this issue, we describe a solution-based method that is capable of characterizing the global flexibilities of nonhelix elements in RNA, provided that such elements are flanked by helix (e.g., bulges, internal loops, or branches). The "phased tau ratio" method is based on the principle that, for RNA molecules possessing two variably phased bends, the relative birefringence decay times depend on the flexibility of each bend, not simply the mean bend angles. The method is used to examine the overall flexibility of the yeast tRNAPhe core (as unmodified transcript). In the presence of magnesium ions, the tRNA core is not significantly more flexible than an equivalent length of RNA helix. In the absence of divalent ions, the tRNA core gains flexibility under conditions where its secondary structure is likely to be largely preserved. The phased tau ratio approach should be broadly applicable to nonhelix elements in both RNA and DNA and to protein-nucleic acid interactions.