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PubMed1996

The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity.

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the Km for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

Amino Acid Sequence↗
PubMed1990

Assignments of the iminoproton resonances of Bombyx mori tRNA(UCCGly) and the comparison of its structure and stability with those of tRNA(GCCGly).

Most of the iminoproton resonances in the 1H-NMR spectrum of Bombyx mori tRNA(UCCGly) have been assigned by the sequential NOEs. Any peak which indicates the presence of the tertiary GC base pair between the D and T loops could not be detected. The effects of temperature and the addition of magnesium ions and spermine on the 1H-NMR spectrum of this tRNA were examined. From the temperature change, it was found that the acceptor stem and the D stem in Bombyx mori tRNA(UCCGly) are equally stable even in the absence of magnesium, which is different from tRNA(GCCGly) where the D stem is not so stable.

Animals↗
PubMed1996

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Archaea↗
PubMed2002

Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation.

A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

Antigens, Viral↗
PubMed1996

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.

Anticodon↗
PubMed2006

Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in lizards.

Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.

Animals↗
PubMed1974

Enzymatic methylations: III. Cadaverine-induced conformational changes of E. coli tRNA fMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation.

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.

Adenosine↗
PubMed2002

p38 is essential for the assembly and stability of macromolecular tRNA synthetase complex: implications for its physiological significance.

Mammalian tRNA synthetases form a macromolecular complex with three nonenzyme factors: p43, p38, and p18. Here we introduced a mutation within the mouse p38 gene to understand its functional significance for the formation of the multi-tRNA synthetase complex. The complex was completely disintegrated by the deficiency of p38. In addition, the protein levels and catalytic activities of the component enzymes and cofactors were severely decreased. A partial truncation of the p38 polypeptide separated the associated components into different subdomains. The mutant mice showed lethality within 2 days of birth. Thus, this work provides the first evidence, to our knowledge, that p38 is essential for the structural integrity of the multi-tRNA synthetase complex and mouse viability.

Amino Acyl-tRNA Synthetases↗
PubMed2002

An RNA complex of the HIV-1 A-loop and tRNA(Lys,3) is stabilized by nucleoside modifications.

The HIV transcription initiation complex involves a putative interaction between the primer tRNA anticodon and a conserved A-rich loop in the HIV genome. Surface plasmon resonance was used to demonstrate that the hypermodified nucleosides in the tRNA anticodon stem loop (ASL) stabilize RNA-RNA interactions in a model for the anticodon/A-loop complex. tRNA ASL hairpins with the modifications of Escherchia coli tRNALys and human tRNALys,3 each form stable complexes. Partially modified tRNA ASLs bind the A-loop hairpin with lesser affinity, and it was found that the modifications of the bacterial and mammalian tRNAs make distinct contributions toward stabilizing the RNA complex. One model for the anticodon/A-loop RNA complex that is consistent with the known modification effects on tRNA structure and function is that of complementary tRNAs, as seen for the published crystal structure of tRNAAsp.

Adenosine↗
PubMed2000

Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs.

Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell.

Catalysis↗
PubMed1994

Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation.

The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

We have done a systematic study on the contribution of the single-stranded NCCA end (where N is any nucleotide) to the stability of the aminoacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' terminus derived from the aminoacyl stem of Escherichia coli tRNA(Ala) and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' terminus noticeably stabilizes the duplex in a sequence-dependent manner. Though the largest contribution to the stability gain due to the ACCA end is provided by the first dangling 3' nucleotide, the influence of even the fourth nucleotide is measurable. The nature of the N73 discriminator base influences the stem structure and stability, which may be important for the recognition of tRNA by aminoacyl-tRNA synthetase. The stepwise attachment of the nucleotides to the 3' tail improves the stacking of the unpaired bases over the helix stem. Hence, the ACCA end appears to be structured. Replacing Mg2+ with Mn2+ causes broadening of certain imino proton peaks in the NMR spectrum, indicating a specific divalent metal ion binding site in the vicinity of the major identity element of the duplex (G3-U70) that is required for its recognition by the Ala-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Another cut for lysine tRNA: application of the hyperprocessing reaction reveals another stabilization strategy in metazoan lysine tRNAs.

Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).

Animals↗
PubMed1976

Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells.

Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure.

tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.

Adenosine Triphosphate↗
PubMed1987

A single base change in the extra-arm of yeast mitochondrial tyrosine tRNA affects its conformational stability and impairs aminoacylation.

The mitochondrial temperature-sensitive mutation tsm-8 maps on a 1.8 kb HpaII fragment of mitochondrial DNA (mt DNA) which contains genes for tRNA(Ala), tRNA(Ile) and tRNA(Tyr). The phenotype of this mutation is, among multiple pleiotropic defects, a temperature-induced reduction of mitochondrial translation. DNA sequencing of the HpaII fragment from the wild type and mutant tsm-8 revealed a single transversion from T to A in position 56 of the mutant tRNA(Tyr) gene. This nucleotide change disrupts a base pairing in the long extra arm of the tRNA cloverleaf. Revertants of the tsm-8 mutant restore correct base pairing in the extra arm by a second-site mutation in the tRNA(Tyr) gene. Analysis of the tRNA(Tyr) transcripts revealed that neither transcription nor processing of the tRNA is affected in the mutant. However, the base alteration destabilizes the conformation of the tRNA and affects its charging parameters. At the non-permissive temperature, the Michaelis-Menten constant of the mitochondrial tyrosyl-tRNA synthetase for the mutant tRNA is increased over 20-fold when compared to the wild-type tRNA. As a consequence, mitochondrial protein synthesis is drastically reduced at the restrictive temperature. Moreover, synthesis of apocytochrome b and of cytochrome oxidase subunit 3 is decreased relative to the other mitochondrially synthesized polypeptides.

Adenine↗
PubMed1997

The origin of the genetic code and protein synthesis.

A model for a parallel evolution of the genetic code and protein synthesis is presented. The main tenet of this model is that the genetic code, that is, a correspondence between nucleotide and amino-acid coding units, originated from sequence-specific interaction between abiotically synthesized polynucleotides and polypeptides. A sequence-specific binding between oligonucleotides and oligopeptides is supported by experimental findings. Moreover, it is parsimonious enough to be consistent with the relatively simple chemistry of a primordial environment. Proximity between peptides and RNA increased the rate of formation of ester bonds between them. This lead to the accumulation of sequence-specific polypeptide-polynucleotide pairs, that is, of primordial-loaded tRNA. Condensation of short polypeptides into longer products could be catalyzed by a sequence-specific juxtaposition of loaded tRNA over complementary RNA, originating the core of protein synthesis. The accumulation of useful encoded products, for example, catalysts for tRNA loading (primordial aminoacyl-tRNA synthetases) or stabilizers of tRNA-mRNA interactions (primordial ribosomes), permitted the subsequent evolution of protein synthesis and of the genetic code to their mature form. This occurred via a parallel reduction in length of the interacting polynucleotides and polypeptides. Thus, it maintained the correct reading frame of mRNA from the preceding stages of evolution.

Evolution, Molecular↗