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At least 37 records · Page 2Linked to original sources
PubMed1977

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 2. Species containing a large variable loop.

The number of base pairs in the solution structure of several class III D3VN tRNA species from E. coli has been determined by analyzing the number of low-field (-15 to -11 ppm) proton resonances in their nuclear magnetic resonance spectra at 360 MHz. Contrary to previous reports indicating the absence of tertiary resonances, all the spectra exhibit the expected number of secondary base pair resonances plus approximately ten extra resonances derived from tertiary base pairs in the three-dimensional folding of these molecules. The possible origins of some of these tertiary resonances are discussed; none of the spectra exhibits the characteristic resonance of the 8-14 tertiary base pair seen in class I D4V5 tRNA spectra.

Binding Sites↗
PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed1977

Initial stages of the thermal unfolding of yeast phenylalanine transfer RNA as studied by chemical modification: the effect of magnesium.

The early stages of thermal unfolding of the tertiary structure of yeast tRNAPhe have been followed, in the presence and absence of Mg2+, by measuring changes in the chemical accessibility of the bases uracil and guanine. The reagent used in these studies is 1-cyclohexyl 3-[2-morpholino(4)-ethyl]carbodiimide methotosylate. 32P-labelled tRNA was used so that the points of modification could be examined with ribonuclease digestion and established fingerprinting techniques. Two regions of protection of Mg2+ have been found. One is within the oligonucleotide U8-A-m2G10 and the other is in the vicinity of residue U-59. The tertiary interactions and the D stem are the most readily melted parts of the teritary structure. In the absence of Mg2+ the region of U-59 is the first part of the tertiary structure to become accessible to the reagent. This is closely followed by the opening up of the 'wobble' G-U base pair in the aminoacyl stem. Most of the triple interactions in the augmented D helix are also disrupted early in the melting. The region of intricate interactions between the invariant G-G part of the D loop and the T-psi-C-G loop contains the most stable set of tertitary structure interactions.

Base Sequence↗
PubMed2026

tRNA methylation: functional insights and epitranscriptomic regulation.

tRNAs, one of the most conserved and abundant RNAs, are central components of protein synthesis, transferring genetic information from DNA to proteins through a precise base-pairing mechanism. Post-transcriptional modifications of tRNAs by tRNA modifying enzymes are essential for maintaining their normal physiological functions, including methylation, isomerization and glycosylation. tRNA methylation, particularly 1-methyladenosine (m1A), 5-methylcytidine (m5C), and 7-methylguanosine (m7G), are among the most abundant and diverse types of post-transcriptional modifications of tRNA, which promote the stability of tRNA secondary and tertiary structures and allow for proper translation. In addition, tRNA methylation affects the production and function of tsRNA (tRNA-derived small RNA), small fragments of RNA that further regulate gene expression and protein synthesis. In our review, we discuss the relevant biological functions of tRNA methylation, including tRNA stability, protein translation, and tsRNA biogenesis.

RNA, Transfer↗
PubMed2026

Effect of Tertiary Lymphoid Structures on Immune Cell Infiltration in the Tumor Microenvironment and Prognosis in Lung Adenocarcinoma.

Tertiary lymphoid structures (TLSs) modulate immune responses in various solid tumors, but their comprehensive role in lung adenocarcinoma (LUAD) remains unclear. In this study, we analyzed RNA-seq data from 539 LUAD patients in The Cancer Genome Atlas (TCGA) and microarray data from 223 samples from the Gene Expression Omnibus (GEO, GSE13213, and GSE37745). TLS signatures were evaluated via unsupervised consensus clustering based on 12 chemokine transcriptome signatures. The relationships between TLS and clinical characteristics, tumor microenvironment (TME) cell infiltration, and prognosis were assessed using ESTIMATE and CIBERSORT. A prognostic model was established using LASSO regression and validated with external datasets. Additionally, H&E and IHC analyses were performed to explore associations between intratumoral TLS density, immune-related molecular expression, and patient prognosis in LUAD. Consensus clustering of the TCGA cohort revealed two distinct LUAD patient clusters according to TLS abundance. Cluster 1 exhibited greater immune cell infiltration, more favorable prognosis, and increased expression of immune checkpoint molecules. We developed a prognostic model comprising eight survival-associated genes that act as independent prognostic factors for patient survival. H&E/IHC analyses revealed that TLS density-regardless of pathological stage-was associated with better prognosis; higher intratumoral TLS density/proportion was also related to more favorable outcomes. IHC confirmed that survival-associated genes (CD5, HLA-DMB, and P2RY13) are independent prognostic indicators in LUAD. Our study demonstrated the close relationship between TLS signatures and an active immune microenvironment, highlighting their potential as independent prognostic indicators in LUAD.

Humans↗
PubMed1979

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↗
PubMed1977

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↗
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↗
PubMed1978

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↗
PubMed1978

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↗
PubMed1977

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↗
PubMed2026

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↗
PubMed1977

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↗
PubMed1978

A calorimetric investigation of melting of tRNAAsp from brewer's yeast.

The thermodynamics of tRNAAsp unfolding was studied using a precision scanning microcalorimeter. The overall heat of melting was found to be about 55 J/g irrespective of the ionic strength and magnesium activity. The analysis of complex melting curves obtained in the absence of Mg2+ reveals four successive two-state transitions. The first was identified as the cooperative melting of the tertiary structure and the D region and the others as the melting of individual helical arms.

Aspartic Acid↗
PubMed1977

[Gene duplication and protein evolution. Case of aminoacyl-t-RNA synthetases].

Aminoacyl-tRNA synthetases are a functionally homologous group of enzymes which catalyse the first step of protein synthesis. The accumulation of data on the oligomeric structures of these enzymes has revealed a wide diversity in the sizes and organization of the protomers. This is not consistent with the idea of a family with homologous primary and tertiary structures. Howewer recent studies have shown that all these enzymes may have evolved from a common ancestor through gene duplication and fusion which has led to extensive repeating sequences in the corresponding subunits.

Amino Acid Sequence↗