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PubMed1979

Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance.

In order to elucidate the functional role of the modified uridines at position 54 of tRNA, the 270 MHz high-field proton NMR spectra of methionine tRNAs from E. coli, from a mutant thereof, and from T. thermophilus, containing ribothymidine, uridine and 2-thioribothymidine, respectively, have been measured as a function of temperature. A comparison of the NMR melting profiles of the minor nucleosides from these tRNAs shows that the melting temperature of uridine containing tRNA is 6 degrees C lower than that of the wild type tRNA whereas that of the 2-thioribothymidine tRNA is 7 degrees C higher than that of the wild type tRNA. These results, therefore, demonstrate that these modifications serve for stabilization of the tertiary structure of tRNA.

Drug Stability↗
PubMed2002

Transition state stabilization by the N-terminal anticodon-binding domain of lysyl-tRNA synthetase.

Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC ) catalyzes aminoacylation of tRNA(Lys) with l-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA(Lys). B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser(1)-Pro(144)) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys(151)-Lys(493)). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction.

Catalytic Domain↗
PubMed1997

Structural studies on tRNA acceptor stem microhelices: exchange of the discriminator base A73 for G in human tRNALeu switches the acceptor specificity from leucine to serine possibly by decreasing the stability of the terminal G1-C72 base pair.

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (aaRS) is crucial to the maintenance of translational fidelity. The discriminator base A73 in human tRNALeuis critical for its specific recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance and converts it into a serine acceptor in vitro . Two RNA microhelices of 24 nt length that correspond to the tRNALeuacceptor stem and differ only in the discriminator base were synthesized: a wild-type tRNALeumicrohelix, where nt 21 corresponds to the discriminator base position 73, and an A21G mutant microhelix. To investigate whether different identities of both tRNAs are caused by conformational differences, NMR and UV melting experiments were performed on both microhelices. Two-dimentional NOESY spectra showed both microhelices to exhibit the same overall conformation at their 3'-CCA ends. Thermodynamic analysis and melting behaviour of the base-paired imino protons observed by NMR spectroscopy suggest that the A21G (A73G in tRNA) exchange results in a decrease of melting transition cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA) base pair. Furthermore, the fact that this 3'-terminal imino proton is more solvent-exposed at physiological temperature might be another indication for the importance of the stability of the terminal base pair for specific tRNA recognition.

Adenine↗
PubMed1975

Initiation and transcription of a set of transfer ribonucleic acid genes in vitro.

Using RNA polymerase purified from Escherichia coli, DNA isolated from the bacteriophage T4, and a bacterial supernatant fraction containing the necessary processing enzymes, a set of transfer RNAs can be formed in vitro. To characterize the site or sites of initiation of this tRNA transcription, rifampicin-resistant complexes of RNA polymerase, DNA, and either ATP (UTP and CTP) or GTP (UTP and CTP) were formed, and tRNA was transcribed from these stabilized sites. It is concluded that transcription of the entire set is initiated by ATP. To study the transcription of the tRNAs, the time sequence of the appearance of individual species was determined during synchronous transcription of a preformed RNA polymerase-DNA complex. The appearance of three RNA species is found to be consistent with the sequential transcription of a large polycistronic cluster; the order and distances, inferred from the times of transcription, are as required by the existing gene map. It is concluded that the initiation of tRNA transcription can occur, without accessory factors, with the insertion of ATP at a single or a few closely spaced sites, and that the tRNAs encoded by the bacteriophage T4 are present in a single operon.

Chromosome Mapping↗
PubMed2005

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics.

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu.GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.

Anti-Bacterial Agents↗
PubMed2006

Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNALeu(UUR)) gene.

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation.

Cell Proliferation↗
PubMed1997

Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.

The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

Amino Acid Sequence↗
PubMed1996

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine.

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Amino Acids↗
PubMed1998

Host mutations (miaA and rpsL) reduce tetracycline resistance mediated by Tet(O) and Tet(M).

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N6-(4-hydroxyisopentenyl)-2-methylthio-adenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

Anti-Bacterial Agents↗
PubMed2002

Selection of tRNA by the ribosome requires a transition from an open to a closed form.

A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity.

Anti-Bacterial Agents↗
PubMed1986

Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine.

Conformational and dynamic properties of the anticodon loop of yeast tRNAPhe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3' side. The influence of Mg2+, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s2UUC of E. coli tRNA2Glu were investigated. Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes (tau) and rotational relaxation times (tau R) we conclude that wybutine occurs in various structural states: one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (tau = 6 ns, tau R = 19 ns), an unstacked conformation where the base can freely rotate (tau = 100 ps, tau R = 370 ps) and an intermediary state (tau = 2 ns, tau R = 1.6 ns). Under biological conditions, i.e. in the presence of Mg2+ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA2Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3'-5' stack.

Anticodon↗
PubMed1987

Binding of DNA hairpins to an assembler-strand as part of a primordial translation device.

A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Only n-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.

Base Sequence↗
PubMed1992

The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB.

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".

Base Sequence↗
PubMed2005

Stressing out over survival: glutamine as an apoptotic modulator.

INTRODUCTION: The amino acid glutamine (GLN) has received considerable attention as a potential therapeutic adjuvant in critical illness and in improving postoperative clinical outcomes. Most studies on the role of GLN in cellular physiology have historically focused on its anabolic roles in specific cell types and its contribution to growth in cancer cells. However, an emerging body of work that examines the consequences of GLN deprivation on cellular survival and gene expression has constructed a new paradigm for this amino acid, namely, that limited extracellular GLN supplies modulate stress and apoptotic responses. METHODS: A survey of the scientific literature was conducted on GLN in cell survival signaling and apoptosis. Work from our laboratory in liver cancer cells also was included in this review. RESULTS: Most studies on this topic have used mammalian cell lines derived from the gut, immune system (including hybridomas), and various cancers. GLN limitation, even in the presence of an adequate glucose supply, impacts stress-related gene expression, differentially modulates receptor-mediated apoptosis, and directly elicits apoptosis through signaling mechanisms and caspase cascades that are specific to cell type. To date, GLN transporters, cellular hydration, glutaminyl-tRNA synthetase, ATP levels, mRNA stability, and glutathione economy have been variably implicated in GLN-dependent survival signaling. CONCLUSION: The cell type-specific mechanisms underlying the regulatory role of GLN in cell survival continue to unfold at a steady pace through in vitro studies. These results have collectively provided testable hypotheses for further in vivo studies into their physiological relevance during GLN "nutritional pharmacology."

Apoptosis↗
PubMed1975

Allosteric mechanism for codon-dependent tRNA selection on ribosomes.

We suggest that the interaction between a codon and its cognate tRNA induces conformational changes in the tRNA. We further suggest that sites on the ribosome preferentially bind tRNA in those conformations which require proper matching of codon and anticodon. According to this model, the codon functions as an allosteric effector which influences the conformation at various sites in the tRNA. This is made possible by the ribosome, which we suggest traps tRNA molecules in those conformation states that maximize the energy difference between cognate and noncognate codon-anticodon interactions. Studies of the interactions between tRNA molecules and their cognate codons in the absence of the ribosome have suggested that triplet-triplet interaction between codon and anticodon is far too weak to account for the specificity of the tRNA selection mechanism during protein synthesis. In contrast, we suggest that such affinity measurements do not adequately describe the interaction between a codon and its cognate tRNA. Thus, such experiments can not detect conformational changes in the tRNA, and, in particular, those stabilized by the ribosome.

Allosteric Regulation↗
PubMed1983

Structural features required for the binding of tRNATrp to avian myeloblastosis virus reverse transcriptase.

The basis of the specific binding of tRNATrp by avian myeloblastosis virus reverse transcriptase was studied by chemical and enzymatic modification of the RNA. Binding does not depend on recognition of the tryptophan anticodon since molecules cleaved in the anticodon are stably bound by the enzyme. Modification of pseudouridine residues in the tRNA destroys binding to reverse transcriptase. These results are consistent with a model in which reverse transcriptase-tRNATrp interaction occurs not at the anticodon, but at regions in the tRNA which contain or are stabilized by pseudouridine residues.

Animals↗
PubMed1979

1H NMR studies on the conformational characteristics of 2-thiopyrimidine nucleotides found in transfer RNAs.

The molecular conformations of naturally occurring 2-thiopyrimidine nucleosides (5-methylaminomethyl-2-thiouridine, 5-methoxycarbonylmethyl-2-thiouridine and 2-thiocytidine) and 5'-mononucleotides (5-methylaminomethyl-2-thiouridine 5'-monophosphate and 2-thiocytidine 5'-monophosphate) in 2H2O solution were elucidated by analyses of the proton NMR spin-coupling constant, nuclear Overhauser effect, and lanthanide-induced shifts and relaxation enhancements. As monomers, these nucleotides are almost exclusively in the 3E-gg-anti form, even in the absence of ordinary stabilizing factors of this form; i. e., base-stacking and base-pairing interactions with other nucleotide units. This inherent conformational rigidity of the 2-thiopyrimidine units probably contributes to stability of the conformation of tRNA.

Escherichia coli↗