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Results for “RNA, Transfer, Val”

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PubMed1986

Mosquito mitochondrial transfer RNAs for valine, glycine and glutamate: RNA and gene sequences and vicinal genome organization.

We report the sequences of 3 transfer RNAs from mosquito (Aedes albopictus) mitochondria, those for valine (anticodon UAC), glutamic acid (anticodon UUC) and glycine (anticodon UCC), as well as sequences for the corresponding genes and for some neighboring mitochondrial genes. TRNAval is notable for its high level of psi, tRNAglu for its low level of G and C, and tRNAgly is notable in that it appears as two species widely separated in gel electrophoresis, differing only in modification status. TRNAglu is the first sequenced insect mitochondrial tRNA that would be expected to engage in U.R wobble (where U is a modified U in the first position of the anticodon, and R is G or A in the third position of codons), if the insect system followed the modified wobble rules proposed for mammalian and fungal mitochondria; and the sequence determined does fit the proposal. The gene for tRNAval follows immediately that for 12S ribosomal RNA. The gene for tRNAglu occurs in a cluster of 6 tRNA genes that is separated from the gene for tRNAgly by a short reading frame. Features of the DNA sequences are discussed with reference to Drosophila, and mammalian, mitochondrial genome organization.

Aedes↗

PubMed1998

Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918.

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.

Animals↗

PubMed1992

Escherichia coli tRNA(Asp) recognition mechanism differing from that of the yeast system.

Various tRNA transcripts were constructed to study the identity elements of Escherichia coli tRNA(Asp). Base substitutions from G34 to U34 at the first position of the anticodon, and from U35 to A35 at the second, severely impaired the aspartate charging activity. The activity was also decreased, but in a more moderate fashion, by base changes at G2-C71, C36 and C38. Identity nucleotides of tRNA(Asp) are distributed in a different fashion between E. coli and yeast, which occur at the second base pair of the acceptor stem, G10-U25 base pair in the D-stem and 3' half of the anticodon loop.

Anticodon↗

PubMed1988

Is translation inhibited by noncognate ternary complexes?

We studied the influence of an error-prone isoacceptor (tRNALeu4), as well as an intermediate (tRNALeu2) and a weak (tRNAVal) competitor of tRNAPhe on the poly(Phe) synthesis rate. Even at very high excess concentrations of these noncognate ternary complexes there was no significant effect on the translation rate. Our result argues against the assertion that in vivo translation is slowed down by noncognate tRNA and favours the hypothesis that the incorrect ternary complex concentrations are too low to saturate the ribosomes in vivo.

Binding, Competitive↗

PubMed1991

Fluorine-19 NMR studies of the thermal unfolding of 5-fluorouracil-substituted Escherichia coli valine transfer RNA.

19F NMR spectroscopy was used to monitor the thermal unfolding of E. coli tRNAVal labeled by incorporation of 5-fluorouracil (FUra). With rising temperatures, resonances in the 19F NMR spectrum of (FUra)tRNAVal gradually shift towards the central region of the spectrum and merge into a single broad peak above 85 degrees C. FU55 and FU12 are the first to shift, beginning at temperatures below 40 degrees C, which suggests that the initial steps of thermal denaturation of tRNAVal involve disruption of the tertiary interactions between the D- and T-arms. The acceptor stem and the FU64-G50 wobble base pair in the T-stem are particularly stable to thermal denaturation. A temperature-dependent splitting of the 19F resonance assigned to FU64, at temperatures above 40 degrees C, suggests that the T-arm of (FUra)tRNAVal exists in two conformations in slow exchange on the NMR time scale.

Base Sequence↗

PubMed1994

The role of the CCA sequence of tRNA in the peptidyl transfer reaction.

Peptidyl transfer is a key step in the process of protein biosynthesis. To examine the role of the universal CCA terminal sequence of tRNA in the process of peptidyl transfer, various mutant transcripts of Escherichia coli valine tRNA were constructed. Peptidyl transferase activity, monitored by the 'fragment reaction' with a slight modification, was decreased by mutation at any one base of CCA. The effect of mutation was moderate in the UCA, CUA and CCG mutants. Replacement of A76 by a pyrimidine nucleotide, or replacement of either C74 or C75 by a purine nucleotide caused a marked decrease in the activity. These findings suggested that the universal CCA terminus of tRNA makes a functional interaction with ribosomal RNA by base-pairing.

Acetylation↗

PubMed1988

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging.

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

Anticodon↗

PubMed1992

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.

Acylation↗

PubMed1989

Interactions of Escherichia coli SO-187 tRNA(IVal) with Bacillus stearothermophilus valine-tRNA synthetase studied by 13C-NMR.

Uracil isotopically labelled with 13C at C4 and C5 has been incorporated into nucleic acids of the Escherichia coli uracil auxotroph, SO-187. [4,5-13C]uracil-labeled tRNA(IVal) was isolated and purified. 13C longitudinal relaxation times measured at 67.8 MHz demonstrated that the C5 dipole caused a 20-50% increase in the C4 relaxation. Interactions of this tRNA with valine-tRNA synthetase (VTS) purified from Bacillus stearothermophilus were established by 13C-NMR. Specific spectral changes were seen at 4-thiouridine, ribothymidine and pseudouridine of the 'bend' in the three-dimensional structure, and particularly at the uridine-5-oxyacetic acid in the wobble position of the anticodon. Thus, the protein seems to be in contact along the entire tRNA molecule, including the anticodon loop.

Amino Acyl-tRNA Synthetases↗

PubMed1994

Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA.

Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods. In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications.

Base Composition↗

PubMed1988

A family of non-allelic tRNA(ValGUU) genes from the cellular slime mold Dictyostelium discoideum.

A haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 14 non-allelic gene copies coding for a tRNA(ValGUU). The structure, genomic organization, and expression of these genes have been analyzed in relation to stages of the developmental cycle. So far, 13 tRNA(ValGUU) genes have been isolated and characterized. All genes contain identical mature tRNA-coding regions, and consequently identical gene internal promoter elements. However, different genes differ with respect to their 5'- and 3'-flanking regions, although a certain degree of sequence conservation seems apparent. Different members of this tRNA gene family appear to be randomly dispersed along the seven D. discoideum chromosomes, and not clustered at any one genomic location. In vivo expression of individual genes was studied in yeast. All but one tRNA(ValGUU) gene are actively transcribed, though with different efficiencies. There is also evidence that not all of these tRNA genes are constitutively transcribed in Dictyostelium throughout the developmental cycle. One characteristic primary transcript can only be detected in cells of the late preaggregation phase, whereas growing cells, cells in the stationary phase or cells harvested 4 h after the onset of development do not seem to carry this transcript. This product seems to be transcribed from a gene of an unusual structure. Although this particular gene has not yet been isolated, it can be predicted from the sequence of the cDNA synthesized from primary transcription products of this putative gene, that it is composed of nt 1-54 of a 3'-truncated tRNA(ValGUU) gene linked to a bona fide tRNA(ValGUU) gene.

Alleles↗

PubMed1989

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: proposed mechanism and significance of tRNA gene dispersion.

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.

Animals↗

PubMed1995

Characterization of oligonucleotides and nucleic acids by mass spectrometry.

The continued refinement of two recent methods for producing gas-phase ions, electrospray ionization and matrix-assisted laser desorption ionization, has resulted in new techniques for the rapid characterization of oligonucleotides by mass spectrometry. Using commercially available instruments, molecular mass measurements at the 20-mer level, with errors less than 2 Da, can now be made routinely in less than 15 min. Progress has also been achieved in the development of mass spectrometry for rapid sequencing of oligonucleotides smaller than 25 residues.

Base Sequence↗

PubMed2006

Translocation of a tRNA with an extended anticodon through the ribosome.

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

Anticodon↗

PubMed2004

Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences.

Advanced frogs (Neobatrachia) are usually divided into two taxa, Ranoidea (the firmisternal frogs) and Hyloidea (all other neobatrachians). We investigated phylogenetic relationships among several groups of Hyloidea using 12S and 16S rRNA mitochondrial gene sequences and tested explicit relationships of certain problematic hyloid taxa using a sample of 93 neobatrachians. Parsimony, maximum likelihood, and Bayesian inference methods suggest that both the Ranoidea and Hyloidea are well-supported monophyletic groups. We reject three hypotheses using parametric bootstrap simulation: (1) Dendrobatidae lies within the Ranoidea; (2) The group containing Hylidae, Pseudidae, and Centrolenidae is monophyletic; and (3) Brachycephalus is part of Bufonidae.

Animals↗

PubMed2004

Endemic ranid (Amphibia: Anura) genera in southern mountain ranges of the Indian subcontinent represent ancient frog lineages: evidence from molecular data.

The geological history of the Indian subcontinent is marked by successive episodes of extensive isolation, which have provided ideal settings for the development of a unique floral and faunal diversity. By molecular phylogenetic analysis of a large set of ranid frog taxa from the Oriental realm, we show that four genera, now restricted to torrential habitats in the Western Ghats of India and the central highlands of Sri Lanka, represent remnants of ancient divergences. None of three other biodiversity hotspots in the Oriental mainland were found to harbour an equivalent level of long-term evolutionary history in this frog group. By unceasingly providing favourable humid conditions, the subcontinent's southern mountain ranges have served as refugia for old lineages, and hence constitute a unique reservoir of ancient ranid endemism.

Animals↗

PubMed2006

Phylogeny of the túngara frog genus Engystomops (= Physalaemus pustulosus species group; Anura: Leptodactylidae).

We present a phylogeny of the Neotropical genus Engystomops (= Physalaemus pustulosus species group) based on sequences of approximately 2.4 kb of mtDNA, (12S rRNA, valine-tRNA, and 16S rRNA) and propose a phylogenetic nomenclature. The phylogeny includes all described taxa and two unnamed species. All analyses indicate that Engystomops is monophyletic and contains two basal allopatric clades. Clade I (Edentulus) includes E. pustulosus and the Amazonian E. petersi + E. cf. freibergi. Clade II (Duovox) includes all species distributed in W Ecuador and NW Peru. Brevivox, a clade of small-sized species is strongly supported within Duovox. Populations of Engystomops pustulosus fall into two well-supported clades, each of which occupies two disjunct portions of the species range. Overall, our phylogeny is congruent with most previous hypotheses. This study is among the few published species-level phylogenies of Neotropical amphibians derived from molecular datasets. A review of the proportion of new species detected by similar studies suggests that the increasing use of molecular techniques will lead to the discovery of a vast number of species of Neotropical amphibians.

Animals↗

PubMed2006

Phylogenetic systematics of the colorful, cyanide-producing millipedes of Appalachia (Polydesmida, Xystodesmidae, Apheloriini) using a total evidence Bayesian approach.

Here, we provide an exemplar-approach phylogeny of the xystodesmid millipede tribe Apheloriini with a focus on genus-group relationships-particularly of the genus Brachoria. Exemplars for the phylogenetic analysis were chosen to represent the maximum breadth of morphological diversity within all nominal genera in the tribe Apheloriini, and to broadly sample the genus Brachoria. In addition, three closely related tribes were used (Rhysodesmini, Nannariini, and Pachydesmini). Morphological and DNA sequence data were scored for Bayesian inference of phylogeny. Phylogenetic analysis resulted in polyphyletic genera Brachoria and Sigmoria, a monophyletic Apheloriini, and a "southern clade" that contains most of the tribal species diversity. We used this phylogeny to track morphological character histories and reconstruct ancestral states using stochastic character mapping. Based on the findings from the character mapping study, the diagnostic feature of the genus Brachoria, the cingulum, evolved independently in two lineages. We compared our phylogeny against prior classifications using Bayes factor hypothesis-testing and found that our phylogenetic hypothesis is inconsistent with the previous hypotheses underlying the most recent classification. With our preferred total-evidence phylogeny as a framework for taxonomic modifications, we describe a new genus, Appalachioria; supply phylogenetic diagnoses of monophyletic taxa; and provide a phylogeny-based classification for the tribe Apheloriini.

Animals↗