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PubMed1993

Acceptor end binding domain interactions ensure correct aminoacylation of transfer RNA.

The recognition of the acceptor stem of tRNA(Gln) is an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNA(Gln).GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNA(1Ser). The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNA(Gln), but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNA(Gln). On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130-->Pro, Glu-131-->Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNA(Glu) more stably than the wild-type enzyme does (Kd is 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.

Amino Acid Sequence↗

PubMed2004

Cofolding organizes alfalfa mosaic virus RNA and coat protein for replication.

Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3' termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3' conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication.

3' Untranslated Regions↗

PubMed1993

Editing corrects mispairing in the acceptor stem of bean and potato mitochondrial phenylalanine transfer RNAs.

Editing is a general event in plant mitochondrial messenger RNAs, but has never been detected in a plant mitochondrial transfer RNA (tRNA). We demonstrate here the occurrence of a tRNA editing event in higher plant mitochondria: in both bean and potato, the C encoded at position 4 in the mitochondrial tRNA(Phe)(GAA) gene is converted into a U in the mature tRNA. This nucleotide change corrects the mismatched C4-A69 base-pair which appears when folding the gene sequence into the cloverleaf structure and it is consistent with the fact that C to U transitions constitute the common editing events affecting plant mitochondrial messenger RNAs. The tRNA(Phe)(GAA) gene is located upstream of the single copy tRNA(Pro)(UGG) gene in both the potato and the bean mitochondrial DNAs. The sequences of potato and bean tRNA(Pro)(UGG) genes are colinear with the sequence of the mature bean mitochondrial tRNA(Pro)(UGG), demonstrating that this tRNA is not edited. A single copy tRNA(Ser)(GCU) gene was found upstream of the tRNA(Phe) gene in the potato mitochondrial DNA. A U6-U67 mismatched base-pair appears in the cloverleaf folding of this gene and is maintained in the mature potato mitochondrial tRNA(Ser)(GCU), which argues in favour of the hypothesis that the editing system of plant mitochondria can only perform C to U or occasionally U to C changes.

Base Sequence↗

PubMed2002

Cysteinyl-tRNA formation and prolyl-tRNA synthetase.

Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process.

Amino Acyl-tRNA Synthetases↗

PubMed2006

The complete mitochondrial genome of the whiting, Merlangius merlangus and the haddock, Melanogrammus aeglefinus: a detailed genomic comparison among closely related species of the Gadidae family.

We determined the first complete mitochondrial DNA (mtDNA) sequences for the whiting (Merlangius merlangus, family Gadidae, order Gadiformes) and the haddock (Melanogrammus aeglefinus, family Gadidae, order Gadiformes). The entire mitogenomes were amplified and sequenced by primer walking using newly designed specific internal primers. Lengths were 16,569 and 16,585 bases for whiting and haddock respectively, lengths which lie within the range of previously reported gadiform sequences from Atlantic cod (Gadus morhua, 16,696 bases) and walleye pollock (Theragra chalcogramma, 16,570 bases). Gene arrangement in both species conformed to the order seen in most vertebrate mitochondrial genomes. We identified a long intergenic spacer located between the tRNA(Thr) and tRNA(Pro) genes (of 100 and 70 bp long for whiting and haddock, respectively), as previously described for other species of the order Gadiformes. Using nucleotide and amino acid divergence data of four complete gadoid mitogenomes (M. merlangius, M. aeglefinus, G. morhua and T. chalcogramma), we examined in detail the relative mtDNA mutation patterns across genes and among Gadidae species and tested for the performance of each protein-coding, transfer RNA and ribosomal RNA gene in depicting the expected phylogeny among the four species, as compared with the whole genome dataset. This comparison may be particularly useful in phylogenetic analyses of such a diverse fish family, as well as for the understanding of the patterns of nucleotide substitution of the mtDNA at low levels of divergence.

Animals↗

PubMed2000

Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg.

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

Base Sequence↗

PubMed1985

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet.

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.

Anticodon↗

PubMed2006

A tryptophan-rich hexapeptide inhibits nucleic acid destabilization chaperoned by the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 exerts critical functions in viral genome replication and virus assembly. Since the recognition of target nucleic acids is required in the initial step of most NC-mediated processes, attempts were made to find small molecules capable of competing with this recognition. In particular, several Trp-rich hexapeptides were recently found to strongly bind RNA sequences targeted by NC. To further validate these peptides as potential anti-NC agents, we studied the ability of Ac-HKWPWW-NH2, taken as a representative, to interfere with the NC chaperone properties required during reverse transcription. Using NMR and steady-state and time-resolved fluorescence spectroscopy, we characterized the structure of Ac-HKWPWW-NH2 as well as its binding to viral sequences such as TAR and PBS involved in the two obligatory strand transfers of reverse transcription. Results show that Ac-HKWPWW-NH2 exhibits an almost symmetric cis-trans equilibrium at the level of the Pro residue where it is structured. The peptide binds both TAR and PBS sequences with low micromolar affinities. The cis-Pro and trans-Pro conformations of the peptide bind with comparable affinities to (-)PBS, mainly through stacking interactions between the Trp residues and the (-)PBS bases. Though all three Trp residues may contribute to the (-)PBS/Ac-HKWPWW-NH2 complex formation, Trp3 and Trp5 residues are the key residues in the complexes with the cis-Pro and trans-Pro conformations, respectively. Moreover, Ac-HKWPWW-NH2 stabilizes cTAR secondary structure and largely inhibits the NC-directed melting of cTAR. This further strengthens the interest of this peptide for deriving modified peptides capable of inhibiting NC and HIV-1 replication.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence of the sheep mitochondrial DNA D-loop and its flanking tRNA genes.

The nucleotide sequence of the sheep mitochondrial DNA displacement-loop (D-loop) region and its flanking tRNA genes has been determined. Several conserved motifs among mammals have been identified along the 1189-bp sequence of the sheep control region: ten termination-associated sequences (TASs) and one conserved sequence block (CSB-1). CSB-2 and CSB-3, which are frequently found in most species, are not present in the sheep D-loop, which shows instead a short direct repeat at their usual localization. A long polypyrimidine tract between CSB-1 and the tRNA(Phe) gene is also present. Furthermore, the sheep mtDNA D-loop region displays tandem repeats in the left domain (adjacent to the tRNA(Pro) gene) comprising three different termination-associated sequences (TAS-5, TAS-6 and TAS-7).

Animals↗

PubMed2002

Replication of avian sarcoma virus in vivo requires an interaction between the viral RNA and the TpsiC loop of the tRNA(Trp) primer.

Reverse transcription in avian sarcoma virus (ASV) initiates from the 3' end of a tRNA(Trp) primer, which anneals near the 5' end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the T psi C loop of the tRNA(Trp) (U5-T psi C). We now demonstrate that this U5-T psi C interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-T psi C interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the T psi C loop of the tRNA(Trp). To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNA(Pro), while simultaneously randomizing the viral RNA U5-T psi C region. After five rounds of infection, the consensus sequence 5'-GPuPuCPy-3' emerged, which is complementary to the 5'-GGTTC-3' sequence found in the T psi C loop of tRNA(Pro). These observations confirm the importance of the U5-T psi C interaction in vivo.

Animals↗

PubMedDate not supplied

Adenovirus-mediated suicide gene transfer to small cell lung carcinoma using a tumor-specific promoter.

The gastrin-releasing peptide (GRP) is expressed in most types of small cell lung carcinoma (SCLC) and the GRP promoter is thought to be potentially useful for tumor-specific expression of the suicide gene in SCLC. We constructed an adenovirus containing the herpes simplex thymidine kinase suicide gene driven by the GRP promoter (AdGRP-TK) and transfected it into GRP-expressing SCLC cells (SBC5) to confer sensitivity to ganciclovir (GCV). After infection with AdGRP-TK, SBC5 cells became more sensitive to GCV in vitro. In nude mice, a subcutaneously-inoculated tumor of SBC5 cells infected with AdGRP-TK in advance regressed completely after intraperitoneal administration of GCV. These results suggest that adenovirus-mediated gene transfer of the suicide gene followed by pro-drug treatment may be applicable to SCLC.

Adenoviridae↗

PubMed1973

Analysis of isoaccepting transfer ribonucleic acid species of Bacillus subtilis: chromatographic differences between transfer ribonucleic acids from spores and cells in exponential growth.

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs.

Acylation↗

PubMed1989

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria.

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.

Amino Acids↗

PubMed2003

Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression.

The Her-2/neu oncogene is overexpressed in approximately 30% of breast and ovarian cancer cases and often indicates a poor prognosis. Therapeutic agents against Her-2/neu have been intensively sought over the past decade. Here we show that small interfering RNA (siRNA) can silence the expression of Her-2/neu in models of human breast or ovarian cancer through retrovirus-mediated transfer of an siRNA against Her-2/neu. Cells infected with retrovirus expressing anti-Her-2/neu siRNA exhibit slower proliferation, increased apoptosis, increased G0/G1 arrest, and decreased tumor growth. Changes in cell cycle-associated factors included decreased levels of phosphatidylinositol 3-kinase, pAkt, and cyclin D1 and increased levels of p27 and phosphorylated retinoblastoma protein. Knockdown of Her-2/neu expression by siRNA is also associated with increased expression of the anti-angiogenic factor thrombospondin-1 and decreased expression of the pro-angiogenic vascular endothelial growth factor, suggesting that Her-2/neu stimulates tumor growth at least in part by regulating angiogenesis. siRNA-mediated gene silencing of Her-2/neu and increasing the expression of thrombospondin-1 may be a useful therapeutic strategy for Her-2/neu-over-expressing breast or ovarian cancer.

Apoptosis↗

PubMed1986

Use of gene transfer approaches to study regulation of expression of opioid peptide genes.

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Adrenal Medulla↗

PubMed1979

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA.

Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the "spacer" sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

Amino Acyl-tRNA Synthetases↗

PubMed1987

Physiologically-induced changes in proopiomelanocortin mRNA levels in the pituitary gland of the amphibian Xenopus laevis.

In the pars intermedia of the pituitary gland of the amphibian Xenopus laevis the level of mRNA encoding proopiomelanocortin (POMC), the precursor protein for alpha-melanophore-stimulating hormone (alpha-MSH), is shown to be dependent on physiological parameters. POMC mRNA levels in the pars intermedia of black-background-adapted Xenopus are much higher than those of white-adapted animals. These physiological changes in POMC mRNA levels are tissue-specific because they were not found in the pars distalis of the pituitary gland. Background transfer experiments revealed that modulation of POMC gene activity is much slower than changes in the secretion of alpha-MSH.

Adaptation, Physiological↗

PubMed2006

Expression of anti-apoptotic protein, Bcl-2, in liver regeneration after a partial hepatectomy.

BACKGROUND: Although Bcl-2 is well known to have anti-apoptotic activities in vitro and in vivo, the role of Bcl-2 relating to liver regeneration remains controversial. The aim of this study was to document the effect of Bcl-2 expression on liver regeneration in rats undergoing a partial hepatectomy. MATERIAL AND METHODS: Adult male Wistar rats (n = 4/group) at 72 h before undergoing a 70% partial hepatectomy (PH) were administered 1 x 10(9) plaque-forming units of adenovirus vector encoding either human Bcl-2 (group 1) or LacZ (group 2) intravenously and were sacrificed at 0, 12 h, and at 1, 2, 3, 7, 14, and 21 days postoperatively. In group 3, normal saline was injected instead of adenovirus vector. Liver regeneration was monitored by measuring the restituted liver mass and proliferating cell nuclear antigen (PCNA) immunostaining. The incidence of apoptosis in the liver was analyzed by the immunohistochemical detection of single-stranded DNA at 14 and 21 days postoperatively. RESULTS: The restituted liver mass showed significantly higher values in group 1 (26.1 +/- 7.2%) than in group 2 (14.7 +/- 6.8%) and 3 (13.6 +/- 5.0%) at 1 day after PH (P < 0.05). The PCNA labeling index was significantly higher in group 1 (47.2 +/- 9.9%) than in groups 2 (19.0 +/- 7.8%) and 3 (19.2 +/- 15.2%) at 1 day after a partial hepatectomy (P < 0.05). The hepatocyte growth factor (HGF) mRNA expression was significantly lower in group 1 than in group 2 at 12 h after PH (P < 0.05). The number of single-stranded DNA-positive cells decreased significantly more in group 1 (5.67 +/- 1.53 positive cells/10 fields per tissue) than those in group 2 (18.33 +/- 7.57 positive cells/10 fields per tissue) at 14 days after PH. CONCLUSIONS: These results thus indicated that an overexpression of anti-apoptotic protein Bcl-2 does not necessarily have an anti-apoptotic effect on liver regeneration but appears to have a pro-proliferative effect in the early phase of liver regeneration.

Adenoviridae↗