Evidence from chemical modification for an unusual tertiary structure of the TpseudouridineC loop in rabbit liver tRNA1Val.
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Alkylation↗
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High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.
In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.
Kinetics↗
RNA-ligant interactions. (I) Magnesium binding sites in yeast tRNAPhe.
X-ray crystallagraphic studies studies indicate that there are at least four site-specifically bound hydrated Mg2+ ions, [Mg(H2O)n]2+, in yeast tRNAPhe. The size and the octahedral coordination geometry, rather than the charge, of [Mg(H2O)N]2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.
Binding Sites↗
Tertiary structure interactions of 7-methylguanosine in yeast tRNA Phe as studied by borohydride reduction.
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Borohydrides↗
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↗
Strong magnesium binding sites in yeast phenylalanine transfer RNA.
To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op....Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m2G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it links the dihydrouridine arm with the extra (V) loop.
Binding Sites↗
Structural domains of transfer RNA molecules.
In this article, we have described various detailed features of the conformation of yeast tRNA(Phe) revealed by recent refinement analysis of x-ray diffraction data at 2.5 A resolution. The gross features of the molecule observed in the unrefined version have been largely confirmed and a number of new features found. The unique role of the ribose 2' hydroxyl groups in maintaining a series of nonhelical conformations in this RNA molecule has become apparent. Many of these features are a direct consequence of the geometry of the ribose phosphate backbone of RNA molecules, and these may also be found in structured regions of other RNA species as well. Special attention has been directed toward two conformational motifs revealed by this analysis. These include the striking similarity between the TpsiC and anticodon hairpin turns in the polynucleotide chain, which are stabilized by the participation of uridine in the U turn. In addition, there is frequent occurrence of an arch conformation in the polynucleotide chian which is stabilized by hydrogen bonds from 2' hydroxyl residues to phosphate groups across the base of the arch. The importance of the 2' hydroxyl interactions in defining tertiary structure is illustrated by the fact that, in the nonhelical regions, almost half of the ribose residues are involved in O2' hydrogen-bonding interactions which stabilize the conformation of the molecule.
Anticodon↗
4-Thiouridine, a built-in probe for structural changes in transfer RNA.
The luminescence of an aqueous solution of 4-thiouridine was compared with its emission when forming part of the polynucleotide chain of tRNA. In both cases excitation into the last absorption band at 335 nm yields a weak emission in the 520--550 nm region. However, while in aqueous solution this emission has a lifetime of approximately 240 ns, it increases in native tRNA to tau congruent to 6.6 mus. Oxygen and Cl- ions quench the thiouridine emission efficiently in aqueous solution while Na+ and Mg2+ ions have no influence on it. On the other hand thiouridine which forms part of a tRNA molecule is quite insensitive to Cl- ions and to O2 while its emission is greatly enhanced by Na+ and Mg2+ ions. From these salt effects as well as from data on the temperature dependence of the emission yield and the decay curve, it is concluded that the site of the thiouridine residue is very well protected within the tertiary structure of tRNA. Both permanent changes in the secondary and in the tertiary structures of the polynucleotide as well as dynamic conformation changes can be observed by following the emission characteristics of its thiouridine residue.
Binding Sites↗
A study of the thermal unfolding of Escherichia coli phenylalanine transfer RNA by chemical modification at elevated temperatures.
Escherichia coli tRNAPhe was modified by 3 M sodium bisulphite pH 6.0 for 24 h in the temperature range 25 degrees C (x 5 degrees C) to 55 degrees C and in the absence of added magnesium ions. The sites and extents of conversion of cytidines to uridine occurring at each temperature were determined by fingerprinting. The new sites of cytidine modification found at higher reaction temperatures were assumed to arise from breakage of secondary and tertiary structure hydrogen bonds involving cytidine residues. From these data, we conclude that hydrogen bonds within the 'complex core' of the tRNA (including the base pairs G-10 . C-25, C-11 . G-24 and C-13 . G-21 within the dihydrouridine stem and the tertiary structure base pair G-15 . C-48 melt at a lower temperature than the tertiary structure hydrogen bonds between G-19 in the dihydrouridine loop and C-56 in the TpsiC loop.
Base Sequence↗
1H NMR studies of transfer RNA III: the observed and the computed spectra of the hydrogen-bonded NH resonances of baker's yeast transfer-RNA Phe.
The hydrogen-bonded NH resonances of Baker's yeast tRNAphe in H2O solution with Mg++ have been measured by a 360 MHz spectrometer at 23 degrees C. Totally, fifteen peaks and one shoulder can be resolved which represent 25 +/- 1 protons. Based on the refined atomic coordinates of the tRNAphe in the orthorhombic crystal, on the recent advances in the distance dependence of the ring-current magnetic field effects and on the adopted values for the isolated hydrogen-bonded NH resonances, a computed spectrum consisting of 23 protons was constructed. A quantitative comparison by computer was made between the computed spectrum and the spectrum simulated from the observed spectrum. These two spectra are closely similar but not identical. We suggest that the conformation of yeast tRNAphe in aqueous solution is closely similar but not identical to that found in the crystal, especially in the T psi C region and D region. Also the NH resonances in 3-4 proposed hydrogen bonds (most likely for tertiary structure) may exchange very rapidly in aqueous solution.
Binding Sites↗
Thermal unfolding of yeast glycine transfer RNA.
In the present investigations the molecular unfolding of yeast tRNA(Gly) has been studied by a combination of nuclear magnetic resonance spectroscopy, melting techniques, and relaxation kinetics. From these studies the following pathway of unfolding was found. In a coupled melting transition the tertiary, the DHU, and the anticodon structure are disrupted. This is followed by the melting of the acceptor arm, while the T psi C arm, which only contains G-C pairs, melts out last. Interestingly, during the first melting transition a new structure not belonging to the original cloverleaf structure is formed. The thermodynamic and kinetic parameters of the melting transitions were determined and are discussed in relation to earlier work. The present nuclear magnetic resonance (NMR) experiments as well as earlier studies show that the ring current calculations based on the cloverleaf structure provide a good first-order interpretation of the NMR spectra of tRNA.
Base Sequence↗
High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.
Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.
Binding Sites↗
Effect of magnesium and polyamines on the structure of yeast tRNAPhe.
The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.
Magnesium↗
A study of secondary and tertiary solution structure of yeast tRNA(Asp) by nuclear magnetic resonance. Assignment of G.U ring NH and hydrogen-bonded base pair proton resonances.
The 270-MHz spectra of yeast tRNA(Asp) in H2O solutions containing Mg2+ show clearly resolved resonances in the region from -15 to -9.5 ppm. Resonances between -15 and -11.5 ppm from the hydrogen-bonded protons of the acceptor stem and anticodon arm decrease in intensity with increasing temperature and disappear by 75 degrees C. Simultansously, four well-resolved resonances between -11.2 and -10.3 ppm also decrease in intensity and disappear. Because of this behavior and their positions these resonances have been assigned to the four ring NH protons of G.U base pairs 5 and 30 in the acceptor stem and anticodon arm which are thereby shown not to be hydrogen bonded by normal Watson-Crick hydrogen bonds. The five G.C base pair resonances of the T psi C arm remain visible above 70 degrees C after all other resonances have disappeared. The high-temperature tRNA spectrum agrees well with that of the isolated T psi C hairpin and CCA half-molecule fragments, each of which contains the same five hydrogen-bonded proton resonances. The root-mean-square error between the observed and calculated resonance positions for the hydrogen-bonded base pair protons of these three arms is 0.19 ppm. The dihydrouridine stem is expected to have two A.U Watson-Crick base pairs and no B.C base pairs. However, it does not contribute any hydrogen-bonded resonances to the nuclear magnetic resonance (NMR) spectrum below -11.5 ppm. This suggests that even at 35 degrees C this helix is not hydrogen bonded in a normal manner. In the region below -11.4 ppm there are three additional proton resonances melting earlier than the rest which cannot be assigned to a particular helix of the cloverleaf. We suggest that these resonances arise from hydrogen-bonded protons involved in stabilizing tertiary structure.
Binding Sites↗
Refining a giant virus lineage: a novel order unifying Mamonoviridae and "Manesviridae," unveiled by the discovery of furtivovirus.
UNLABELLED: The evolutionary origins and taxonomic framework of giant viruses related to the family Mamonoviridae and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba Vermamoeba vermiformis as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of Nucleocytoviricota viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family Mamonoviridae. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within Nucleocytoviricota. IMPORTANCE: Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family Mamonoviridae and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.
Giant Viruses↗
Nonintercalative binding of ethidium bromide to nucleic acids: crystal structure of an ethidium--tRNA molecular complex.
X-ray diffraction studies at 4.5-A resolution on crystals of a complex of ethidium bromide and yeast phenylalanine tRNA reveal a nonintercalative model of binding of the ethidium within the tertiary structure. This is contrary to the expected interacalative binding to the double-helical regions.
Binding Sites↗
Interactions of yeast tRNAPhe with ribosomes from yeast and Escherichia coli. A fluorescence spectroscopic study.
The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichia coli was studied by stead-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA-ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNAPheEtd was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TpsiC loop.
Binding Sites↗
Rate of tritium labeling of specific purines in relation to nucleic acid and particularly transfer RNA conformation.
The kinetics of the incorporation of tritium into the C-8 positions of purine units in nucleic acids has been studied. The polymers investigated include poly(A), poly(A): poly (U) duplex, a double-stranded viral RNA, tRNA, and DNA. In the random coil state, the kinetics of incorporation of tritium into the purine sites of the polymers are identical with those for the corresponding purine mononucleotides. When the nucleic acids are in their native conformations, however, the purine labeling rates are reduced below that expected for the free mononucleotides. The magnitude of the effect is remarkably dependent upon the particular nucleic acid. For example, at 37 degrees C the purines in double-stranded DNA label at a rate two- to threefold slower than the corresponding mononucleotides, but in a double-stranded viral RNA, a 30- to 40-fold effect is found. The data suggest a strong influence of microscopic helix structure on the rate of tritium incorporation. First-order rate constants for the exchange of tritium into specific purine sites in yeast tRNAPhe were also determined. This was done by partially labeling the nucleic acid in tritiated water, and subsequently removing free and loosely bound tritium. Under conditions where exchange-out does not occur, the nucleic acid was digested with specific nucleases; chromatographic separation then enabled specific activities of purines from specific sites to be obtained. The rate constants for these sites show a large variation. They are markedly reduced for those residues occurring in cloverleaf helical sections and, in certain cases, for those known from crystallographic data to be involved in tertiary interactions. As examples of bases that can participate in tertiary interactions, the crystal structures show A14 and G15 in special base-pairing arrangements. Both purines (A14 and G15) occur in single-stranded sections of the cloverleaf; both show markedly reduced C-8 hydrogen-exchange rates. On the other hand, rate constants for bases and regions known to be on the outside of the moleculesuch as the anticodon loop and the 3' terminusāre perturbed the least. In one instance, a base in the dihydrouridine loop believed to be involved in tertiary interactions, according to crystallographic studies, incorporates tritium as if it were relatively unperburbed by the tRNA structure. The structural interactions of this base may be partially or completely broken at 37 degrees C in solution.
Base Sequence↗