SEARCH · PubMed Health

Results for “RNA, Transfer, Pro”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources

PubMed1993

Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa.

RNA polymerase II is a multisubunit enzyme composed of two large subunits of molecular weight in excess of 100,000 and a collection of 8-10 smaller subunits. The largest subunit, designated IIa, contains at its carboxyl terminus a highly repetitive domain consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Extensive phosphorylation within this COOH-terminal domain (CTD) gives rise to subunit IIo which has a markedly reduced mobility in SDS-polyacrylamide gel electrophoresis (PAGE) relative to subunit IIa. Recent evidence suggests that RNA polymerase IIA, containing an unphosphorylated CTD, is involved in preinitiation complex assembly, whereas RNA polymerase IIO is involved in elongation. Consequently, CTD phosphorylation is thought to occur after RNA polymerase II has bound to the promoter by a protein kinase that stably associates with the preinitiation complex. We present here the partial purification and characterization of two distinct CTD kinases from a HeLa cell transcription extract. These CTD kinases, designated CTDK1 and CTDK2, are fractionated by chromatography on Mono Q. CTDK1 catalyzes the incorporation of approximately 33 pmol of phosphate/pmol of calf thymus RNA polymerase subunit IIa, almost exclusively on serine. CTDK2 catalyzes the incorporation of approximately 50 pmol of phosphate/pmol of calf thymus subunit IIa, predominantly on serine; appreciable phosphate transfer onto threonine is also observed. Phosphorylation by CTDK2, but not CTDK1, results in a complete mobility shift in SDS-PAGE of subunit IIa to the position of IIo. CTDK1 can utilize ATP, dATP, or GTP as phosphate donor, whereas CTDK2 can utilize only ATP or dATP. The apparent Km for ATP is 30 microM for CTDK1 and 60 microM for CTDK2. CTDK1 and CTDK2 also differ in their protein substrate specificity. CTDK1 phosphorylates casein whereas CTDK2 does not. Neither kinase phosphorylates phosvitin or histone H1 to an appreciable extent. CTDK1 and CTDK2 do not appear to be related to cdc2 kinases as determined by their inability to phosphorylate H1 and their failure to react with antibodies directed against the cdc2 kinase. These results establish that a partially fractionated HeLa transcription extract contains two distinct CTD kinases that differ in their nucleotide requirements and in their patterns of CTD phosphorylation.

Amino Acid Sequence↗

PubMed1996

The sequence of 12.8 kb from the left arm of chromosome XIV reveals a sigma element, a pro-tRNA and six complete open reading frames, one of which encodes a protein similar to the human leukotriene A4 hydrolase.

We have determined the nucleotide sequence of a 12.8 kb fragment from the left arm of chromosome XIV carried by the cosmid 14-16d. An analysis of the sequence reveals the presence of a sigma element, a pro-tRNA gene and eight open reading frames, six of which are complete. All of the eight open reading frames correspond to new genes. Of the eight new genes, two show strong similarities to a pair of new genes from chromosome IX, suggesting an ancestral duplication, and one gene encodes a protein similar to mammalian leukotriene A4 hydrolase.

Amino Acid Sequence↗

PubMed1990

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters.

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.

Alleles↗

PubMed1986

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs.

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Base Sequence↗

PubMed1988

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

Avian Sarcoma Viruses↗

PubMed1989

High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product.

We have observed that proteins, such as human tissue-type plasminogen activator, pro-urokinase or gp41 of human immunodeficiency virus, which have a high content of rare codons in their respective genes, are not readily expressed in Escherichia coli. Furthermore induction of these heterologous genes leads to growth inhibition and plasmid instability. Supplementation with tRNA(AGA/AGG(Arg)) by cotransfection with the dnaY gene, which supplies this minor tRNA, resulted in high-level production with greatly improved cell viability and plasmid stability.

Base Sequence↗

PubMed2004

Implication of bax in Xenopus laevis tail regression at metamorphosis.

Apoptosis is fundamental to normal vertebrate development. A dramatic example of postembryonic development involving apoptosis is tail regression during amphibian metamorphosis. Earlier studies led us to propose a functional role for the pro-apoptotic protein Bax in tadpole tail regression. However, its physiological relevance has never been analyzed. We have now cloned a cDNA encoding Xenopus laevis bax (xlbax) and used in vivo gene transfer in tail muscle to analyze the effects of xlbax overexpression. Furthermore, by using an antisense strategy in a similar experimental paradigm, xlbax antisense mRNA was shown to block the apoptotic effects of xlbax and protect against apoptosis in metamorphosing tadpoles. Our results suggest that xlbax is a regulator of muscle fiber death in the regressing tail during metamorphosis.

Amino Acid Sequence↗

PubMed1984

Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP.

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.

Escherichia coli↗

PubMed1996

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.

Animals↗

PubMed1989

Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides.

We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M1) protein responsible for down-regulating endogenous transcription of A/WSN/33 influenza virus. A more refined map of the antigenic determinants of the M1 protein was obtained by binding of epitope-specific monoclonal antibodies (MAbs) to chemically cleaved fragments. Epitope 2-specific MAb 289/4 and MAb 7E5 reverse transcription inhibition by M1 protein and react with a 4-kilodalton cyanogen bromide fragment extending from amino acid Gly-129 to Gln-164. Anti-idiotype serum immunoglobulin G prepared in rabbits immunized with MAb 289/4 or MAb 7E5 mimicked the action of M1 protein by inhibiting transcription in vitro of influenza virus ribonucleoprotein cores. This transcription-inhibition activity of anti-MAb 7E5 immunoglobulin G and anti-MAb 289/4 immunoglobulin G could be reversed by MAb 7E5 and MAb 289/4 or could be removed by MAb 7E5-Sepharose affinity chromatography. Transcription of influenza virus ribonucleoprotein was inhibited by one of three synthetic oligopeptides, a nonodecapeptide SP3 with an amino acid sequence corresponding to Pro-90 through Thr-108 of the M1 protein. Of all the structural proteins of influenza virus, only NP and M1 showed strong affinity for binding viral RNA or other extraneous RNAs. The 4-kilodalton cyanogen bromide peptide (Gly-129 to Gln-164), exhibited marked affinity for viral RNA, the binding of which was blocked by epitope 2-specific MAb 7E5 but not by MAbs directed to three other epitopes. Viral RNA also bound strongly to the nonodecapeptide SP3 and rather less well to anti-idiotype anti-MAb 7E5; these latter viral RNA-binding reactions were only slightly blocked by preincubation of anti-MAb 7E5 or SP3 with MAb 7E5. These experiments suggest the presence of at least two RNA-binding sites, which also serve as transcription-inhibition sites, centered around amino acid sequences 80 through 109 (epitope 4?) and 129 through 164 (epitope 2) of the 252 amino acid M1 protein of A/WSN/33 influenza virus. A hydropathy plot of the M1 protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an alpha-helix-forming intervening hydrophobic domain.

Animals↗

PubMed1997

Multiple mitochondrial tRNA(Leu[UUR]) mutations associated with infantile myopathy.

We have identified a cluster of mitochondrial tRNA(Leu[UUR]), mutations in a severe case of infantile myopathy. There were A to G transitions found at mtDNA positions 3259, 3261, 3266 and 3268. These point mutations change the anticodon arm and the anticodon UAA, normally found in tRNA(Leu[UUR]), to UGA which is the one of the tRNAs(Ser[UCN]). This is the first anticodon alteration described in this tRNA. Another swap straight to the anticodon of tRNA(Pro) alone was recently described in a less severe case. Until now infantile myopathies have not been attributed to defined mtDNA alterations. This study reports for the first time mtDNA point mutations causing this early onset of a mitochondrial disorder. The apparent homoplasmy of these mutations and especially the location in the anticodon must be considered lethal, if the child would not have been respirated for 5 years from its birth.

Animals↗

PubMed2000

Synthesis of cysteinyl-tRNA(Cys) by a genome that lacks the normal cysteine-tRNA synthetase.

Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).

Acylation↗

PubMed1983

Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells.

Animals↗

PubMed2002

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.

Angiogenesis Inhibitors↗

PubMed2006

tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition.

The structure, phylogeny and in vivo function of the base pair formed between nucleotides 32 and 38 of the tRNA anticodon loop are reviewed. The A32-U38 pair, which is highly conserved in tRNA2(Ala) and sometimes observed in tRNA2(Pro), was recently found to decrease the affinity of tRNAs to the ribosomal A site relative to other 32-38 combinations. This suggests that the role of 32-38 pair is to tune the tRNA affinity in the A site to a uniform value. New experiments presented here show that the U32C mutation in tRNA1(Gly) increases its affinity to the cognate codon and to codons with third position mismatches in the A site. This suggests that one reason for uniform tRNA binding to evolve was to avoid incorrect codon recognition.

Anticodon↗

PubMed2006

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species.

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.

Algorithms↗

PubMed2004

RGM and its receptor neogenin regulate neuronal survival.

Repulsive guidance molecule (RGM) is an axon guidance protein that repels retinal axons upon activation of the neogenin receptor. To understand the functions of RGM-neogenin complexes in vivo, we used gene transfer technology to perturb their expression in the developing neural tube of chick embryos. Surprisingly, neogenin over-expression or RGM down-expression in the neural tube induces apoptosis. Neogenin pro-apoptotic activity in immortalized neuronal cells and in the neural tube is associated with the cleavage of its cytoplasmic domain by caspases. Thus neogenin is a dependence receptor inducing cell death in the absence of RGM, whereas the presence of RGM inhibits this effect.

Animals↗

PubMed1995

Codon usage, genetic code and phylogeny of Dictyostelium discoideum mitochondrial DNA as deduced from a 7.3-kb region.

We have sequenced a region (7,376-bp) of the mitochondrial (mt) DNA (54 kb) of the cellular slime mold, Dictyostelium discoideum. From the DNA and amino-acid sequence comparisons with known sequences, genes for ATPase subunit 9 (ATP9), cytochrome b (CYTB), NADH dehydrogenase subunits 1, 3 and 6 (ND1, ND3 and ND6), small subunit rRNA (SSU rRNA) and seven tRNAs (Arg, Asn, Cys, Lys, f-Met, Met and Pro) have been identified. The sequenced region of the mtDNA has a high average A + T-content (70.8%). The A + T-content of protein-genes (73.6%) is considerably higher than that of RNA genes (61.3%). Even with the strong AT-bias, the genetic code employed is most probably the universal one. All seven tRNAs are able to form typical clover leaf structures. The molecular phylogenetic trees of CYTB and SSU rRNA suggest that D. discoideum is closer to green plants than to animals and fungi.

Amino Acid Sequence↗