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At least 289 records · Page 16Linked to original sources
PubMed1993

Role of dimerization in yeast aspartyl-tRNA synthetase and importance of the class II invariant proline.

Cytoplasmic aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) from yeast is, as are most class II synthetases, an alpha 2 dimer. The only invariant amino acid in signature motif 1 of this class is Pro-273; this residue is located at the dimer interface. To understand the role of Pro-273 in the conserved dimeric configuration, we tested the effect of a Pro-273-->Gly (P273G) substitution on the catalytic properties of homo- and heterodimeric AspRS. Heterodimers of AspRS were produced in vivo by overexpression of their respective subunit variants from plasmid-encoded genes and purified to homogeneity in one HPLC step. The homodimer containing the P273G shows an 80% inactivation of the enzyme and an affinity decrease for its cognate tRNA(Asp) of one order of magnitude. The P273G-mutated subunit recovered wild-type enzymatic properties when associated with a native subunit or a monomer otherwise inactivated having an intact dimeric interface domain. These results, which can be explained by the crystal structure of the native enzyme complexed with its substrates, confirm the structural importance of Pro-273 for dimerization and clearly establish the functional interdependence of the AspRS subunits. More generally, the dimeric conformation may be a structural prerequisite for the activity of mononucleotide binding sites constructed from antiparallel beta strands.

Aspartate-tRNA Ligase↗
PubMed1995

Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.

Animals↗
PubMed1990

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

Animals↗
PubMed2005

Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex.

Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Synthesis of cysteinyl-tRNACys by a prolyl-tRNA synthetase.

The question of how cysteinyl-tRNA(Cys) is synthesized in organisms that lack a canonical cysteinyl-tRNA synthetase (CysRS) is an important open question in understanding protein synthesis. The prolyl-tRNA synthetase (ProRS) of wide ranging organisms has the ability to mis-activate cysteine without editing. This raises the question of whether the mis-activated cysteine can be charged to tRNA(Cys) to synthesize the correctly matched cys-tRNA(Cys), which may serve as an option in organisms that lack the conventional CysRS. Despite intense searches, such an activity has not been found. Here we show that the ProRS of the radiation-resistant bacterium Deinococcus radiodurans has the ability to charge cysteine to tRNA(Cys) and to retain the cognate pair cys-tRNA(Cys) stably without editing. The cysteinylation by D. radiodurans ProRS is not the major route for synthesis of cys-tRNA(Cys), however, because the organism encodes the conventional CysRS. Nonetheless, the synthesis of cys-tRNA(Cys) in vitro is important and it is unlike previously documented mis-charging of Methanocaldococcus jannaschii ProRS, which synthesizes cys-tRNA(Pro) but not cys-tRNA(Cys). We suggest that the cysteinylation activity of D. radiodruans ProRS may, in the presence of additional factors, offer one option to organisms that lack the conventional CysRS to synthesize cysteinyl-tRNA(Cys) in vivo.

Amino Acids↗
PubMed1990

Codon recognition mechanisms in plant chloroplasts.

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA(Ala)(U*GC) cp tRNA(Pro)(U*GG) and cp tRNA(Arg)(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that 'U:U and U:C wobble' mechanisms can operate to allow the reading of these codons by cp tRNA(Leu)(UAm7G).

Amino Acid Sequence↗
PubMed2005

Functional identification of catalytic metal ion binding sites within RNA.

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Animals↗
PubMed1995

Heterologous expression as a tool for gene identification and analysis.

These days, genome research mainly concerns the accumulation of sequence data and their theoretical interpretation based on analogies to known genes, proteins and structures. However, a final identification of gene function can only be verified by experimental data. One step in this process is the expression of the isolated gene in pro- and eukaryotes. In this article we will review some of the basic features of expression in Escherichia coli and mammalian cells that are relevant to the design of expression experiments. Emphasis is put on the first instance of attaining a high enough level of expression in order to be able to detect the cellular effects or to isolate the product of the transferred gene.

Animals↗
PubMed2000

Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication.

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).

3T3 Cells↗
PubMed1982

Adsorption chromatography of nucleic acids on siliconized porous glass.

Bovine liver tRNA, E. coli rRNA or calf thymus DNA adsorbed on siliconized porous glass in 5M NaCl-0.01M Tris-HCl at pH 7.6 were chromatographed by the lowering of NaCl concentration on the columns. tRNAs were eluted at 5-3M NaCl and were separated in the eluting order of Pro, Val, Ile, Thr, Ser and Phe tRNA. RNA species in crude rRNA were also separated. A half part of calf thymus DNA passed through the column in 5M NaCl-0.01M Tris-HCl and the residual adsorbed was eluted at 2-0M NaCl. The DNA adsorbed is larger than the DNA passed through. Recovery of those nucleic acids from the column was 83-100%.

Animals↗
PubMed1999

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA.

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.

Adenocarcinoma↗
PubMed1993

Direct stimulation of the pituitary by transfer of activated leucocytes.

Some strains of rat develop arthritis, and have profoundly elevated adrenocorticotropin (ACTH) and corticosterone, following intradermal injection of an adjuvant containing heat-killed mycobacteria. Transfer to syngeneic recipients of immune spleen (IS) cells taken from arthritic rats 14 days after injection of the adjuvant, but not of non-immune cells, causes increased circulating ACTH and increased pituitary proopiomelanocortin (POMC) mRNA. Transfer of immune cells does not transfer the disease, but does protect recipients from subsequent challenge with the adjuvant. In these immune-protected rats, the secondary immune response raises ACTH and POMC to levels similar to those seen in arthritic rats. These data show that endogenous inflammatory mediators have direct actions on the neuroendocrine system.

Adrenocorticotropic Hormone↗
PubMed2003

Gene therapy for ocular neovascularization: a cure in sight.

Recent advances in the field of ocular gene transfer have led researchers pursuing treatment for age-related macular degeneration and diabetic retinopathy to turn to gene therapy for the answer. Viral vector-mediated delivery of both anti-angiogenic proteins and molecules that inhibit the eye's endogenous pro-angiogenic factors has successfully diminished the pathology of ocular diseases in rodent models. A gene-therapy solution to the debilitating blindness caused by ocular neovascularization may be on the horizon.

Adenoviridae↗
PubMed2003

Early regulation of hypothalamic arcuate nucleus CART gene expression by short photoperiod in the Siberian hamster.

Cocaine- and amphetamine-regulated transcript (CART) mRNA is expressed in a number of hypothalamic nuclei including the arcuate nucleus (ARC). An increase in CART gene expression in the ARC of juvenile female Siberian hamsters (Phodopus sungorus) 14 days after transfer to short photoperiod at weaning and prior to major divergence of body weight trajectory in this seasonal mammal implicates CART in the induction of programmed weight change. In the current series of experiments, elevated CART mRNA in short photoperiod juvenile female animals relative to long photoperiod controls was apparent throughout the caudal-rostral extent of the ARC after 14 days, but was not observed when short photoperiod exposure was limited to 4-7 days. Elevated CART gene expression was also observed in juvenile males 14 days after transfer to short photoperiod at weaning, in adult female hamsters 14 days after transfer to short photoperiod and in adult male hamsters 21 days after transfer to short photoperiod. There were no consistent trends in expression levels of other energy balance-related genes with these relatively short duration photoperiod manipulations, suggesting that CART may be involved in short photoperiod-programmed body weight regulation.

Animals↗
PubMed1988

A 5 kb intergenic region containing ori1 in the mitochondrial DNA of Saccharomyces cerevisiae is dispensable for expression of the respiratory phenotype.

A mutant mitochondrial DNA of Saccharomyces cerevisiae strain R 0.54 has been shown to have a 5 kb deletion which removes the ori1 sequence and its surrounding intergenic sequences between the pro tRNA and 15 S rRNA genes. The deleted DNA has been sequenced and is found to be flanked by two 11 bp direct repeats. The deletion mutant displays a normal phenotype in non-fermentable media. This implies that the intergenic sequence is not required for expression of the respiratory phenotype.

Base Sequence↗
PubMed1993

Modulation of the activity of angiogenin by mutagenesis at Asp-116.

Substitution of Asn, Ala or His for Asp-116 in angiogenin increases its ribonucleolytic activity towards tRNA and, at least in the case of His, its ability to induce blood-vessel formation (Harper, J.W. and Vallee, B.L. (1988) Proc. Natl. Acad. Sci. USA 85, 7139-7143). Six additional Asp-116 mutants have been examined to further probe the basis for this phenomenon. Substitution of Val, Lys, Glu, or Ser increases activity towards tRNA 2-, 4-, 9- and 16-fold, respectively, whereas substitution of Trp and Pro leads to 2- and 10-fold decreases, respectively. Similar changes are seen in activity towards rRNA. Studies of base-cleavage specificity towards dinucleotide substrates (NpN') reveal a change in preference for G vs. A at the N' position when Ser replaces Asp-116 and a diminished preference for C vs. U at the N position. The Pro, Lys and Glu mutants have essentially unchanged angiogenic activity. The results demonstrate that the principal effect of replacing Asp-116 in angiogenin is to modulate enzymatic activity, possibly through an effect on His-114, and suggest that Asp-116 plays a role in controlling specificity.

Amino Acid Sequence↗
PubMed2006

Involvement of SMAD4, but not of SMAD2, in transforming growth factor-beta1-induced trophoblast expression of matrix metalloproteinase-2.

Matrix metalloproteinases (MMPs) play crucial roles in extravillous trophoblast invasion. In the present study, we examined the possible role of Smad4 and Smad2 in transforming growth factor (TGF)-beta1-induced MMP-2 expression, using the well-established invasive extravillous trophoblast cell line HTR-8/SVneo. Recombinant sense Smad4 or Smad2 retroviral vectors were constructed by inserting full-length Smad4 or Smad2 cDNA into pLXSN retroviral vector. Stable PT67 packaging cell clones were isolated and viral supernatants were used to infect HTR-8/SVneo cells. Effects of retroviral expression of Smad4 and Smad2 on TGF-beta1-regulated MMP-2 expression were assessed by semi-quantitative reverse transcription-polymerase chain reaction and gelatin zymography. The results showed that over-expression of Smad4 augmented MMP-2 mRNA abundance and the secretion of pro-MMP-2, and mimicked the inductive effect of TGF-beta1 on the production of MMP-2. However, retrovirus-mediated sense Smad2 gene transfer had no effect. These findings suggest that Smad4, but not Smad2, mediates TGF-beta1-induced MMP-2 expression in invasive extravillous trophoblasts.

Blotting, Western↗