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At least 1,279 records · Page 71Linked to original sources
PubMed1985

Analysis of modification-dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA.

The conformation of the anticodon loop of Escherichia coli tRNAArg was investigated. It is shown that the structure of the anticodon loop is influenced by the base composition of the anticodon stem, and the natural modification of the nucleoside residue 32 in the anticodon loop. The structural effects detected by analysis of the accessibility of the anticodon loop to nuclease S1 could be correlated with the ability of different Arg-tRNAArg species to suppress frame-shifting during translation of MS2 RNA.

Anticodon↗
PubMedDate not supplied

Incorporation of two anthraquinonylmethyl groups into the 2'-O-positions of oligonucleotides: increased affinity and sequence specificity of anthraquinone-modified oligonucleotides in hybrid formation with DNA and RNA.

Oligonucleotide 15-mers containing one or two anthraquinonylmethyl groups at specified sugar residues have been prepared on an automated DNA/RNA synthesizer by using 5'-O-dimethoxytrityl 2'-O-(2-anthraquinonylmethyl)uridine 3'-O-(2-cyanoethyl)-N, N-diisopropylphosphoramidite. The purification of the modified oligonucleotides was done with denaturing polyacrylamide gel electrophoresis. The base compositions and the presence of anthraquinone group(s) in the oligonucleotides were verified with enzymatic digestion (snake venom phosphodiesterase and alkaline phosphatase) analysis and UV-vis spectral measurements. The UV melting behaviors indicate that all the oligonucleotides with anthraquinone group(s) can bind to both their complementary DNA and RNA in a manner similar to that of the unmodified oligonucleotide. All the oligonucleotides possessing anthraquinone group(s) have higher affinity for both DNA and RNA segments when compared with the unmodified oligonucleotide. The oligomer containing two anthraquinone substituents at sites separated by four nucleotides instead of six exhibits the highest affinity for both the complementary DNA and RNA. The stabilizing effect can be translated into a free energy cost of 7.1 kcal/mol for the DNA hybrid and 3.6 kcal/mol for RNA. It has been shown through mismatch/Tm studies that modification of the oligonucleotide by anthraquinone groups does not alter the sequence specificity in binding to a RNA segment.

Anthraquinones↗
PubMed1973

Ribonucleic acid polymerase in a thermosensitive sporulation mutant (ts-4) of Bacillus subtilis.

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try(-)try(-) (168tt) parental strain were infected with the virulent phage phie at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage phie deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the beta subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.

Bacillus subtilis↗
PubMed2003

Covalent binding of leukotriene A4 to DNA and RNA.

Leukotriene A(4) (LTA(4)) is a highly reactive electrophilic intermediate formed during the biosynthesis of the lipid mediators leukotriene B(4) and leukotriene C(4). Deoxynucleosides were found to react as nucleophiles with LTA(4) in aqueous solutions as assessed by UV spectroscopy and electrospray ionization mass spectrometry. Aqueous solutions of native DNA and RNA were also found to react with LTA(4) as assessed by mass spectrometric analysis of the constituent nucleosides derived from enzymatic hydrolysis of the nucleic acids. The most abundant adducts were observed for guanine- and adenine-containing deoxynucleosides and nucleosides. At neutral pH, these reactions led to an overall modification of deoxyguanosine/guanosine residues in DNA and RNA at 15 +/- 1 adducts/10(7) bases and 230 +/- 20 adducts/10(7) bases, respectively, determined by quantitative assay using stable isotope-labeled LTA(4)-nucleoside adduct. An estimation of the relative reactivity of LTA(4) with each of the purine and pyrimidine bases in DNA and RNA was carried out by comparisons of the mass spectral ion abundance of the different adducts (LTA(4)-dAdo, LTA(4)-dCyd, LTA(4)-Thd, LTA(4)-Ado, LTA(4)-Cyd, and LTA(4)-Urd) to the ion signal of known amounts of LTA(4)-dGuo and LTA(4)-Guo standards. The data were corrected for different mass spectrometric response factors that were experimentally determined for each adduct product. The structures of the two most abundant LTA(4)-Guo products were determined by NMR, UV spectroscopy, and mass spectrometry to be 5-hydroxy,12-[Guo-N(2)-yl]-6,8,11,14-eicosatetraenoic acid. Stimulation of human neutrophils with calcium ionophore led to the covalent modification of DNA within the cell as determined by mass spectrometric analysis of lipophilic nucleosides obtained after hydrolysis of extracted DNA. These observations, combined with the intracellular site of 5-lipoxygenase translocation and LTA(4) biosynthesis at the nuclear envelope, suggest that LTA(4) may have access to DNA and RNA within cells and furthermore modify nucleic acids in situ following the activation of 5-lipoxygenase and initiation of LTA(4) biosynthesis.

Calcimycin↗
PubMed2006

Control of protein expression through mRNA stability in calcium signalling.

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.

3' Untranslated Regions↗
PubMed2004

Phosphorylation of histone H2A inhibits transcription on chromatin templates.

The regulation of gene expression via the histone code has, for the most part, revealed that histone modifications cause the recruitment of adaptor proteins that indirectly regulate the synthesis of RNA. Using purified factors to assemble and modify the chromatin and to transcribe the DNA, we investigated whether modifications of histones may directly impact the RNA polymerase II transcription process. We screened proteins known to modify histones for effects on transcription, and we found that the mitogen- and stress-induced kinase, MSK1, inhibited RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, as naked DNA templates were resistant to the effects of MSK1. We found that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Furthermore, we found that acetylation of histone H3 by the p300 and CREB-binding protein associated factor, PCAF, suppressed the kinase-dependent inhibition of transcription. These results suggest that acetylation of histones may stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1.

Chromatin↗
PubMed1992

The origin and early evolution of nucleic acid polymerases.

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA polymerase beta' subunit and its homologues is discussed. We show that in the DNA-dependent RNA polymerases from the three cellular lineages a very conserved sequence of eight amino acids also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein is present. The optimal conditions for the replicase activity of the avian myeloblastosis virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is also discussed.

Amino Acid Sequence↗
PubMed2006

Microarray tools for deciphering complex diseases.

Individual genetic findings associated with complex diseases are unlikely to fully explain their substantial impact or provide new comprehensive insights into disease pathogenesis. These also lack the comprehensive data much needed for development of new effective drugs in majority of the disease cases in a population. In fact multilevel etiologic factors underlie almost all human diseases, including: environmental causes, epigenetic factors, DNA mutations, amplifications, and deletions, RNA expression levels, protein (translation, post translation modification, localization) and combinations thereof. Each individual might consist of different combinations of these multiple etiologic factors. Integrative evaluation of all these modifications will shed light on the whole identity of the disease and the underlying molecular mechanisms. Until now it was inconceivable to have a full grasp of such a complex etiology. Microarrays enable us to interrogate the individualized various factors (DNA, RNA and protein content) involved in disease state on genome-wide scale simultaneously and expeditiously in single cell or the tissue of interest (Figure 1). The new disciplines of microarray studies in combination hold the promise of effective, current, and comprehensive understanding of complex diseases and may be a good approach for reducing the costs and time lines associated with discovery and efficacy improvement of therapeutic drugs. In the future, through utilizing the colossal amount of microarray data findings, defining the structure, function, and dynamics of entire biological pathways and cellular networks under various physiological states, and the development of robust and efficient methods for analyzing and interpreting high dimensional data, it will be possible to connect combination of experimental results with individualized disease state. This will facilitate precise diagnosis prognosis and therapy.

Alternative Splicing↗
PubMed1989

Zinc release from Xenopus transcription factor IIIA induced by chemical modifications.

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.

Animals↗
PubMed2000

Cancer proteomics: from identification of novel markers to creation of artifical learning models for tumor classification.

Studies of global protein expression in human tumors have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Many changes in gene expression recorded between benign and malignant human tumors are due to post-translational modifications, not detected by analyses of RNA. Proteome analyses have also yielded information about tumor heterogeneity and the degree of relatedness between primary tumors and their metastases. Results from our own studies have shown a similar pattern of changes in protein expression in different epithelial tumors, such as decreases in tropomyosin and cytokeratin expression and increases in proliferating cell nuclear antigen (PCNA) and heat shock protein expression. Such information has been used to create artificial learning models for tumor classification. The artificial learning approach has potential to improve tumor diagnosis and cancer treatment prediction.

Biomarkers, Tumor↗
PubMed2005

Perspectives in spicing up proteomics with splicing.

In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.

Alternative Splicing↗
PubMed2006

DNA methyltransferases: facts, clues, mysteries.

DNA methylation plays a pivotal role during development in mammals and is central to transcriptional silencing. The DNA methyltransferases (DNMTs) are responsible for the generation of genomic methylation patterns leading to gene silencing, but the underlying molecular basis remains largely shrouded in mystery. Here we review our current understanding of the mechanisms by which DNMTs repress transcription and how they are targeted to preferred DNA sequences. Emerging evidence points to an essential and intricate web of interactions between DNMTs and the chromatin environment in which they function. The recent identification of novel transcription factors recruiting the DNMTs may open new avenues of research into the origin of DNA methylation patterns. Thanks to these emerging clues, researchers have begun to lift the veil on the multi-faceted DNMTs, but there remains fascinating work ahead for whoever wants to fully understand DNMTs and their role in the mammalian cell.

Animals↗
PubMed2006

DNA methylation in plants.

DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA sequence identity; therefore, it is mainly associated with CNG and non-symmetrical methylations (rare in animals) in coding and promoter regions of silenced genes. Cytoplasmic viral RNA can affect methylation of homologous nuclear sequences and it maybe one of the feedback mechanisms between the cytoplasm and the nucleus to control gene expression.

Adenine↗
PubMed2000

Biomolecular stability and life at high temperatures.

It is not clear what the upper temperature limit for life is, or what specific factors will set this limit, but it is generally assumed that the limit will be dictated by molecular instability. In this review, we examine the thermal stability of two key groups of biological molecules: the intracellular small molecules/metabolites and the major classes of macromolecules. Certain small molecules/metabolites are unstable in vitro at the growth temperatures of the hyperthermophiles in which they are found. This instability appears to be dealt with in vivo by a range of mechanisms including rapid turnover, metabolic channelling and local stabilisation. Evidence to date suggests that proteins have the potential to be stable at substantially higher temperatures than those known to support life, but evidence concerning degradative reactions above 100 degrees C is slight. DNA duplex stability is apparently achieved at high temperature by elevated salt concentrations, polyamines, cationic proteins, and supercoiling rather than manipulation of C-G ratios. RNA stability seems dependent upon covalent modification, although secondary structure is probably also critical. The diether-linked lipids, which make up the monolayer membrane of most organisms growing above 85 degrees C are chemically very stable and seem potentially capable of maintaining membrane integrity at much higher temperatures. However, the in vivo implications of the in vitro instability of biomolecules are difficult to assess, and in vivo data are rare.

Archaea↗
PubMed1999

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3.

Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3' and 5' 'core' replication origins (promoters) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting 'replicon' can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to approximately 240 nucleotides of sequence from the region immediately upstream of the core 3'-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral 'spacer' sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3'-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons.

Chenopodiaceae↗
PubMed1991

Endonuclease (R) subunits of type-I and type-III restriction-modification enzymes contain a helicase-like domain.

A statistically significant amino acid sequence similarity is demonstrated between the endonuclease (R) subunit of EcoK restriction-modification (R-M) enzyme, and RNA and DNA helicases of the so-called 'DEAD' family. It is further shown that all three known sequences of R subunits of type-I and type-III R-M enzymes contain the conserved amino acid sequence motifs typical of the previously described helicase superfamily II [(1989) Nucleic Acids Res. 17, 4713-4730]. A hypothesis is proposed that these enzymes may exert helicase activity possibly required for local unwinding of DNA in the cleavage sites.

Amino Acid Sequence↗
PubMed1987

Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion.

We have identified a set of Tn5-generated mutants of Rhizobium meliloti on the basis of their failure to form a fluorescent halo under UV light when grown on agar medium containing Calcofluor. These mutations define a new genetic locus we have termed exoH. Alfalfa seedlings inoculated with exoH mutants form ineffective nodules that do not contain intracellular bacteria or bacteroids. Root hair curling is significantly delayed and infection threads abort in the nodule cortex. Analyses of exopolysaccharide secreted by exoH mutants have shown that it is identical to the Calcofluor-binding exopolysaccharide secreted by the exoH+ parental strain except for the fact that it completely lacks the succinyl modification. In vitro translation of total RNA isolated from nodules induced by an exoH mutant has shown that only one of the plant-encoded nodulins is induced, as compared with the 17 nodulins induced by wild-type strains. These observations suggest that succinylation of the bacterial polysaccharide is important for its role(s) in nodule invasion and possibly nodule development.

DNA Transposable Elements↗