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PubMed1990

Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli.

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

Amino Acids↗
PubMed2002

Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation.

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.

Base Sequence↗
PubMed1998

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Base Sequence↗
PubMed1994

Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation.

The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1974

Heterologous mischarging as a means of tRNA fractionation. I. Behaviour of E. coli phenylalanyl-tRNA(1Val) on BD-cellulose.

The chromatographic behaviour of E.coli tRNA(Val) (1), Val-tRNA(Val) (1) and Phe-tRNA(Val) (1) was studied on BD-cellulose columns. At pH 4.0 and 4 degrees C the elution position of Phe-tRNA(Val) (1) was not affected by the presence of absence of Mg(2+), whereas Val-tRNA(Val) (1) was slightly retarded when Mg(2+) was ommited. It is postulated that the amino acid and its nature influence the structure that the aminoacyl-tRNA assumes. Under suitable conditions the heteroaminoacylated Phe-tRNA(Val) (1) eluted significantly later than other tRNAs. This fact showed that heterologous mischarging can be a useful step in tRNA purification methods.

Chromatography, DEAE-Cellulose↗
PubMed2005

Comparison of the global structure and dynamics of native and unmodified tRNAval.

The effects of post-transcriptional modifications on the structure and dynamics of Escherichia coli tRNA(val) were studied by NMR spectroscopy. NMR chemical shift data and residual dipolar couplings were used to show that the local secondary and tertiary structures are very similar in native and unmodified tRNA(val). Rigid body restrained molecular dynamics calculations showed that the global structure of tRNA is unchanged by the post-transcriptional modifications. Deuterium exchange NMR experiments were used to probe the dynamics and flexibility of native and unmodified tRNA(val). A similar set of slowly exchanging (t(1/2) > 3 min) imino protons were observed in both tRNAs, but the rates of exchange for the slowest exchanging imino protons were approximately 20 times faster in unmodified than in native tRNA. These results demonstrate that the dynamics and flexibility of tRNA(val), but not the local or global structure, are significantly affected by post-transcriptional modifications.

Base Sequence↗
PubMed1994

Functional transfer RNAs with modifications in the 3'-CCA end: differential effects on aminoacylation and polypeptide synthesis.

The trinucleotide CCA sequence is present at the 3' terminus of all mature tRNAs. Despite this high degree of conservation, we have been able to prepare in vitro transcripts of Escherichia coli tRNA(Val) with altered 3' termini that are readily aminoacylated and can function in polypeptide synthesis. Replacement of the 3'-terminal adenosine with either cytidine or uridine yields a tRNA(Val) variant that retains almost full aminoacylation activity, having specificity constants (Vmax/Km) 40-50% that of wild-type tRNA(Val). The tRNA(Val) variant with a 3'-terminal guanosine remains fully chargeable but is a poor substrate for valyl-tRNA synthetase, largely as the result of a decrease in the catalytic constant. End-group analysis revealed the absence of adenosine at the 3' end of the tRNA(Val) mutants and identified the nucleotide expected from the sequence of the DNA template as the predominant 3'-terminal residue; Val-cytidine was isolated from the aminoacylated C76 mutant. Val-tRNA(Val) with 3'-CCG is active in poly(U,G)-directed (Val, Phe) copolypeptide synthesis, whereas the tRNA(Val) mutants terminating in 3'-CCC and 3'-CCU, which are readily aminoacylated, are inactive. The differential effects of nucleotide substitution on aminoacylation and polypeptide synthesis suggest that the universally conserved 3'-CCA end of tRNAs is monitored at two or more steps in protein synthesis that have different nucleotide recognition specificities.

Base Sequence↗
PubMed1991

Identity determinants of E. coli tRNAs.

Identity determinants of E. coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts. Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val). Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm.

Codon↗
PubMed2000

Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent "double sieve" mechanism. In this study, we determined the 2.9 A crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate. The analog is bound in a pocket, where Pro(41) allows accommodation of the Val and Thr moieties but precludes the Ile moiety (the first sieve), on the aminoacylation domain. The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val). A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve). Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.

Adenosine↗
PubMed2004

A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome.

Ribosome complexes containing deacyl-tRNA1(Val) or biotinylvalyl-tRNA1(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the tRNA1(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450-5454]. However, in complexes containing deacyl-tRNA1(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA1(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.

Base Sequence↗
PubMed2003

Analysis of processing-defective variants of human tRNA(Val).

Eukaryotic tRNAs are transcribed, processed and exported from the nucleus to the cytoplasm to participate in protein biosynthesis. A variant of human gene for tRNA(Val), HtV4, is not processed neither its 5'- nor its 3'-flanking sequences. By RT-PCR analysis and in situ hybridization, we demonstrated that the HtV4 was constitutively expressed without processing, and localized in the nucleus in HeLa cells.

Base Sequence↗
PubMed1996

Molecular cloning of GAF2, a Schizosaccharomyces pombe GATA factor, which has two zinc-finger sequences.

By low stringency screening of a lambda-Shizosaccharomyces pombe genomic library, we have cloned a GATA factor homologous gene, gaf2+, within a 3.1-kb EcoRI fragment. The gaf2 ORF predicts a protein of M(r) 61 kDa consisting of intronless 564 amino acids corresponding to 1,692 bp. Gaf2 has two zinc-fingers as Urbs1 of Ustilago maydis, whereas most of fungal GATA factors have only one zinc-finger. The separation between two zinc-fingers of Gaf2 is rather long. In addition to gaf2, the sequence analysis revealed a Val-tRNA gene in the 3'-flanking region of gaf2. Northern blot analysis indicated that the gaf2 gene is transcribed constitutively irrespective of the nitrogen source in a medium.

Amino Acid Sequence↗
PubMed1997

Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes.

UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.

Base Sequence↗
PubMed2006

Integration into the phage attachment site, attB, impairs multicellular differentiation in Stigmatella aurantiaca.

Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.

Attachment Sites, Microbiological↗
PubMed1990

An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor.

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

Base Sequence↗
PubMed2000

EBNA-1 sequence variation in Danish and Chinese EBV-associated tumours: evidence for geographical polymorphism but not for tumour-specific subtype restriction.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is consistently expressed in EBV-associated tumours. Recently, EBNA-1 carboxy (C)-terminal sequence variants have been described based on the amino acid signature at codon 487, and designated prototype (P)-ala (identical to prototype B95.8 strain), P-thr, variant (V)-val, V-leu, and V-pro. These studies suggest that certain EBNA-1 variants show selective cell tropism and may be preferentially associated with different EBV-positive malignancies; for example, in contrast to P-ala subtypes, V-val appeared to be restricted to the oral compartment and to be associated with undifferentiated nasopharyngeal carcinoma (NPC). To test the hypothesis that V-val subtypes are restricted in distribution, EBNA-1 variants were investigated in NPC and throat washings (TWs) from a low (Denmark) and a high (China) NPC risk area. For comparison, cases of Hodgkin's disease (HD) were also studied. V-val was found to be the dominant EBNA-1 subtype, not only in Chinese TWs and NPC biopsies, but also in Chinese HD. Furthermore, V-val was not detected in any of the Danish NPC biopsies or TW samples. These findings show that V-val is not associated with NPC, nor is it restricted to the oral compartment, but rather that it represents a dominant Asian EBNA-1 subtype, both in EBV-associated malignancies and in the general population.

China↗
PubMed1990

Genes, variant genes, and pseudogenes of the human tRNA(Val) gene family are differentially expressed in HeLa cells and in human placenta.

Pre-tRNAs(Val) were identified in unfractionated tRNA preparations from HeLa cells and human placenta and their 5' leader structures were deduced from the nucleotide sequences of the corresponding cDNAs. Several of these precursors can be assigned to nine out of the eleven members of the human tRNA(Val) gene family characterized so far, which demonstrates that these gene loci are actively transcribed in vivo. Among the expressed genes there are (a) genes for the two known tRNA(Val) isoacceptor species from human placenta, (b) gene variants that exhibit sequence alterations as compared to conventional genes, and (c) pseudogenes that produce processing-deficient precursors which are not matured to tRNAs. The transcription products of several yet unknown tRNA(Val) genes have also been detected. Furthermore, different expression patterns are observed in the two cell types studied. These data allow for the first time an insight into the in vivo expression of a human tRNA gene family.

Base Sequence↗