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At least 127 records · Page 7Linked to original sources
PubMed2001

A new mutation in the mitochondrial tRNA(Ala) gene in a patient with ophthalmoplegia and dysphagia.

We describe a new mutation in the tRNA(Ala) gene, a T-->C transition at nucleotide position 5628, in a 62-year-old woman with late onset chronic progressive external ophthalmoplegia, dysphagia and mild proximal myopathy. The mutation is heteroplasmic and disrupts a highly conserved A-U base pair within the anticodon stem of the tRNA(Ala). Cytochrome c oxidase-negative fibers harbor a significantly higher level of mutated mtDNA than cytochrome c oxidase-positive fibers. This is the first mutation in the tRNA(Ala) gene which satisfies accepted criteria for pathogenicity.

Base Sequence↗
PubMed1995

Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene.

We find striking similarities in promoter structure and requirements for template commitment on 5S RNA and tRNA genes from silkworms. The promoters are nearly the same size (approximately 160 bp) and include flanking as well as internal sequences. To analyze the factor requirements for 5S RNA transcription complex assembly in a completely homologous system, we have isolated a silkworm fraction that is highly enriched for the 5S RNA-specific transcription factor, TFIIIA. Using this fraction, together with the other silkworm fractions, TFIIIB, TFIIIC, TFIIID and RNA polymerase III, we demonstrate that the requirements for 5S RNA transcription complex assembly are very similar to those previously established for a tRNA(C)(Ala) gene. Specifically, no individual factor fraction is sufficient for commitment of silkworm 5S RNA genes to transcription complex assembly. Rather, combinations of at least three factor fractions are required. Our observation that more than one subset of factors is competent for commitment suggests that silkworm 5S RNA genes further resemble tRNA(C)(Ala) genes in their ability to use multiple pathways for transcription complex formation.

Animals↗
PubMed2000

Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli.

Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

Acylation↗
PubMed2005

Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding.

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA(2)(Ala). This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.

Anticodon↗
PubMed1991

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Animals↗
PubMed1991

Four sites in the acceptor helix and one site in the variable pocket of tRNA(Ala) determine the molecule's acceptor identity.

The structural features that determine tRNA(Ala) acceptor identity have been studied with amber-suppressor tRNAs in Escherichia coli cells. Previous work established that a wobble pair composed of guanosine at position 3 and uridine at position 70 (G3-U70) in the acceptor helix of tRNA(Ala) is a determinant of the molecule's acceptor identity. We show that additional determinants are located at three other sites in the acceptor helix and at one site in the variable pocket of tRNA(Ala). These latter determinants are less important than G3.U70 since their individual alterations in mutants of tRNA(Ala) have smaller degrading effects on the functions of the molecules, and subsets of the determinants, when combined with G3.U70, are sufficient to switch the identities of several other tRNAs to that of tRNA(Ala). Other workers are using fragments of the tRNA(Ala) acceptor helix to study the molecule's acceptor identity. Our demonstration that the variable pocket contributes to tRNA(Ala) acceptor identity means that such fragments do not faithfully replicate the structure-function relationship of the cellular process.

Anticodon↗
PubMed2000

TATA-Binding protein-TATA interaction is a key determinant of differential transcription of silkworm constitutive and silk gland-specific tRNA(Ala) genes.

We have investigated the contribution of specific TATA-binding protein (TBP)-TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA(C)(Ala) gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA(SG)(Ala) gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA(C)(Ala) promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA(C)(Ala) promoter contains two functional TBP binding sequences that overlap, the tRNA(SG)(Ala) promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA(SG)(Ala) promoter since provision of either of the wild-type TATA sequences derived from the tRNA(C)(Ala) promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA(SG)(Ala) gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA(C)(Ala) and tRNA(SG)(Ala) promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA(SG)(Ala) promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.

Animals↗
PubMed2006

The 3' CCACCA sequence of tRNAAla(UGC) is the motif that is important in inducing Th1-like immune response, and this motif can be recognized by Toll-like receptor 3.

In this article, the immunogenicity of tRNA and the recognition of tRNA by Toll-like receptors (TLRs) are analyzed. Analyses of the effects of different tRNA(Ala)(UGC) fragments (tRNA(Ala)1-76 [corresponding to positions 1 through 76], tRNA(Ala)26-76, tRNA(Ala)40-76, tRNA(Ala)62-76, tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70) on the immune responses of hepatitis B surface antigen (HBsAg) were performed with BALB/c mice. Results show that tRNA(Ala)1-76, tRNA(Ala)26-76, tRNA(Ala)40-76, and tRNA(Ala)62-76 adjuvants not only induced stronger T helper (Th) 1 immune responses but also cytotoxic-T-lymphocyte (CTL) responses relative to tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70 adjuvants in HBsAg immunization. A deletion of the D loop (tRNA(Ala)26-76), anticodon loop (tRNA(Ala)40-76), or TpsiC (tRNA(Ala)62-76) loop of tRNA(Ala)(UGC) does not significantly decrease the adjuvant characteristic of tRNA(Ala)(UGC). However a deletion of the 3'-end CCACCA sequence (tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70) of tRNA(Ala)(UGC) significantly decreased the adjuvant characteristic in Th1 and CTL immune responses. Moreover, the recognitions of different tRNA(Ala)(UGC) fragments by TLR3, TLR7, TLR8, and TLR9 were analyzed. Results show that a deletion of the 3' CCACCA sequence of tRNA(Ala)(UGC) significantly decreased the recognition by TLR3. We concluded that the 3' CCACCA sequence of tRNA(Ala)(UGC) is the important motif to induce Th1 and CTL responses and this motif can be effectively recognized by TLR3.

Adjuvants, Immunologic↗
PubMed2006

Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization.

Bombardment of a highly expressed dicot tRNA(ala)(GAC) gene into Zea mays bz-E2 or bz-E5 coleoptiles causes suppression of an Ala(458 )-->Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA(ala)(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA(ala)(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.

Cotyledon↗
PubMed1997

Sequence of DNA 16S/23S spacer region of Leuconostoc oenos (Oenococcus oeni): application to strain differentiation.

Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making. An increasing number of wines are being inoculated with malolactic starters to control the process, and the identification and differentiation of selected strains are now indispensable both for quality control of production and for commercial purposes. In the present work we evaluated the potential use of the intergenic regions of three L. oenos strains for their differentiation. The three 16S/23S rRNA intergenic spacers were amplified in vitro by PCR, and sequences were compared. The spacer sequence was highly conserved in all strains. Inside this spacer, a tRNA-Ala gene containing an 18-bp sequence stretch which is conserved in all tRNA genes was discovered. This sequence, together with random primers, was used for characterization of ten L. oenos strains by PCR.

DNA, Ribosomal↗
PubMed2001

Translocation within the acceptor helix of a major tRNA identity determinant.

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.

Alanine-tRNA Ligase↗
PubMed2004

Suppression of a mitotic mutant by tRNA-Ala anticodon mutations that produce a dominant defect in late mitosis.

Cold-sensitive dominant mutants scn1 and scn2 of Schizosaccharomyces pombe were isolated by their ability to suppress temperature-sensitive cut9-665 defective in an essential subunit (human Apc6/budding yeast Cdc16 ortholog) of anaphase promoting complex/cyclosome (APC/C). APC/C mutants were defective in metaphase/anaphase transition, whereas single scn mutants showed the delay in anaphase spindle elongation at 20 degrees C. The scn mutants lost viability because of chromosome missegregation, and were sensitive to a tubulin poison. To understand the scn phenotypes, mutant genes were identified. Surprisingly, scn1 and scn2 have the same substitution in the anticodon of two different tRNA-Ala (UGC) genes. UGC was altered to UGU so that the binding of the tRNA-Ala to the ACA Thr codon in mRNA became possible. As cut9-665 contained an Ala535Thr substitution, wild-type Cut9 protein was probably produced in scn mutants. Indeed, plasmid carrying tRNA-Ala (UGU) conferred cold-sensitivity to wild-type and suppressed cut9-665 in a dominant fashion. The previously identified scn1(+) (renamed as scn3(+)) turned out to be a high copy suppressor for scn1 and scn2. These are the first tRNA mutants that cause a mitotic defect.

Anaphase↗
PubMed2006

Pure myopathy associated with a novel mitochondrial tRNA gene mutation.

The authors describe a 47-year-old man who presented with proximal muscle weakness, myalgia, elevated creatine kinase, and features of a pure myopathic syndrome in whom they have identified a novel mutation in the mitochondrial tRNA(Ala) gene. This 5591G>A transition is heteroplasmic, segregates with cytochrome c oxidase deficiency in single muscle fibers, and fulfills recognized criteria for pathogenicity. This case exemplifies the wide-ranging clinical spectrum of mitochondrial disease presentations.

Creatine Kinase↗
PubMed1999

Disorder and twin refinement of RNA heptamer double helices.

An RNA helix with seven base pairs which was derived from the acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALA(wt)), as well as a variant, rGGGGCUA.rUAGCCCC (ALA(C70)), in which the single G.U wobble base pair of ALA(wt) was replaced by G.C, crystallize in space group C2. Both non-isomorphic crystal forms display a complex packing pattern, which can be described alternatively as disorder or pseudo-merohedral twinning. The structure of ALA(wt) was determined by SIRAS phasing using an isomorphous iodine derivative, rGGGGCi(5)UA.rUAGCUCC (ALA(I)). All three RNA structures were subsequently subjected to twin refinement in space group P1, using anisotropic thermal displacement parameters at resolutions of 1.16, 1.23 and 1.4 A for ALA(wt), ALA(I) and ALA(C70), respectively. Alternatively, the structure of ALA(wt) was refined in space group C2 assuming twofold disorder of the molecular orientation. The refined structures are of reasonable quality according to all available indicators. There are no systematic differences between the molecular models resulting from twin refinement and disorder refinement.

Base Sequence↗
PubMed2007

Codon reading by tRNAAla with modified uridine in the wobble position.

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.

Base Sequence↗
PubMed2005

A mutation in the anticodon of a single tRNAala is sufficient to confer auxin resistance in Arabidopsis.

Auxin-resistant mutants have been useful for dissecting the mechanisms that underlie auxin-mediated biological processes. Here we report the isolation and molecular characterization of a novel auxin-resistant mutant in Arabidopsis (Arabidopsis thaliana). Like known mutated AUX/IAA transcription factors, the mutant described here displayed dominant resistance to exogenously supplied auxins (sirtinol, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid) and a host of pleiotropic phenotypes, including apical hook deformation, defects in lateral root development, reduced stature, and homozygous lethality. This mutant showed the same sensitivity to the ethylene precursor 1-aminocyclopropane carboxylic acid as wild-type plants, and retained the ability to induce IAA19 expression in response to exogenously supplied indole-3-acetic acid. To our surprise, these phenotypes were not caused by a mutation in an AUX/IAA gene, but rather a mutation in a tRNA(ala) gene in which the anticodon was found changed from CGC to CAC. Such a change results in a tRNA that is charged with alanine but recognizes the second most highly used valine codon in Arabidopsis. Therefore, the observed phenotypes are likely the composite of stochastic mutations of many proteins, including downstream effectors.

Anticodon↗