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At least 415 records · Page 23Linked to original sources
PubMed1972

Structure and function of Escherichia coli formylmethionine transfer RNA: loss of methionine acceptor activity by modification of a specific guanosine residue in the acceptor stem of formylmethionine transfer RNA from Escherichia coli.

The structural requirements of E. coli formylmethionine tRNA for aminoacylation have been examined by chemical modification of the tRNA, followed by separation of the modified molecules into active and inactive components. Photooxidation of tRNA(fMet) at 50 degrees in the presence of methylene blue results in modification of two guanosine (G) residues in the acceptor stem, at positions no. 2 and no. 71 from the 5'-phosphate terminus. Both of these modifications are present in inactive molecules, but only the G residue at position no. 2 is modified in the acceptor stem of active molecules. Loss of methionine acceptance occurs with first-order kinetics, indicating that inactivation by modification of G residue no. 71 is independent of any other modifications taking place under these conditions. The presence of a modified G residue at position no. 2 in the acceptor stem of active photooxidized molecules shows that disruption of normal base-pairing in this region is not sufficient to inactivate tRNA(fMet). These data indicate that the inactivating modification at position no. 71 is lethal due to a specific alteration in the nucleotide base, rather than simply as a result of breaking a hydrogen-bonded base pair in the acceptor stem.

Acylation↗
PubMed2006

Minimum sequence requirements for selective RNA-ligand binding: a molecular mechanics algorithm using molecular dynamics and free-energy techniques.

In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.

Algorithms↗
PubMed2004

RNA sequence elements required for high affinity binding by the zinc finger domain of tristetraprolin: conformational changes coupled to the bipartite nature of Au-rich MRNA-destabilizing motifs.

Tristetraprolin (TTP) binds AU-rich elements (AREs) encoded within selected labile mRNAs and targets these transcripts for rapid cytoplasmic decay. RNA binding by TTP is mediated by an approximately 70-amino acid domain containing two tandemly arrayed CCCH zinc fingers. Here we show that a 73-amino acid peptide spanning the TTP zinc finger domain, denoted TTP73, forms a dynamic, equimolar RNA.peptide complex with a 13-nucleotide fragment of the ARE from tumor necrosis factor alpha mRNA, which includes small but significant contributions from ionic interactions. Association of TTP73 with high affinity RNA substrates is accompanied by a large negative change in heat capacity without substantial modification of RNA structure, consistent with conformational changes in the peptide moiety during RNA binding. Analyses using mutant ARE substrates indicate that two adenylate residues located 3-6 bases apart within a uridylate-rich sequence are sufficient for high affinity recognition by TTP73 (K(d) <20 nm), with optimal affinity observed for RNA substrates containing AUUUA or AUUUUA. Linkage of conformational changes and binding affinity to the presence and spacing of these adenylate residues provides a thermodynamic basis for the RNA substrate specificity of TTP.

Amino Acid Motifs↗
PubMed1992

RNA unwinding by eukaryotic initiation factor 4A and nucleotide modification.

Unwinding of double-stranded RNA by nuclear helicases can lead to modification of adenosine-residues, resulting in inosine. During initiation of protein synthesis the 5' untranslated region of an mRNA is unwound by eukaryotic initiation factors (eIF) -4A and -4B. In this work we investigated the possible nucleotide modification after unwinding by eIF-4A and eIF-4B of in vitro synthesized, labeled RNA. The products of unwinding were analyzed by gel-electrophoresis and, after nuclease digestion, by thin layer chromatography of the mononucleotides. Crude protein fractions unwound the duplex RNA and converted part of the AMP-residues into IMP-residues. However, unwinding by purified factors was not linked to this conversion, the deamination of AMP residues. Concluding, unwinding of RNA during initiation of protein synthesis does not lead to conversion of adenosine into inosine.

Base Sequence↗
PubMed1986

Effect of salts on abortive and productive elongation catalysed by wheat germ RNA polymerase II.

Modification of the ionic conditions in reaction assays containing wheat germ RNA polymerase II and poly(dAT) as template markedly alters the catalytic properties of the transcription complexes. These effects have been studied by measuring the rate of abortive initiation and the extent of productive RNA synthesis. Using combinations of metal ions or various salts, a marked inhibition of abortive initiation was always associated with an increased length of RNA chains. These results are discussed in terms of modulation of the stability of transcription complexes induced by salts or divalent cations. The behavior exhibited by wheat germ RNA polymerase II is also discussed in comparison with previously reported results for procaryotic and eucaryotic RNA polymerases.

Escherichia coli↗
PubMed2006

Cleavage of the siRNA passenger strand during RISC assembly in human cells.

A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.

Gene Silencing↗
PubMed1992

Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

Bacterial Proteins↗
PubMed1996

UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Cell Line↗
PubMed2006

Modifications in Thermus thermophilus 23 S ribosomal RNA are centered in regions of RNA-RNA contact.

Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.

Binding Sites↗
PubMed1984

Stimulation of RNA polymerases from mouse spleen by polyamines and its modification by ammonium sulfate.

Nuclear RNA polymerases Ia, Ib, II and III purified from spleen of Swiss albino mice (Mus musculus) were stimulated significantly by the polyamines, spermine and spermidine, in the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, the optimal stimulating concentrations of both the polyamines for the RNA polymerases were generally diminished and more physiological. 8 mM spermine stimulated both RNA polymerases Ia and II in the presence of sulfate ions, but inhibited both enzymes significantly in the absence of sulfate ions. Stimulation of mouse spleen RNA polymerase II by spermine affects elongation of RNA chains whereas inhibition by spermine affects initiation of RNA synthesis.

Ammonium Sulfate↗
PubMed1983

Early increases in RNA polymerase I activity and 18S and 28S rRNA synthesis in the male rat pituitary after oestradiol treatment.

This article reports the effect of a single injection of 17 beta-oestradiol on RNA synthesis, in the male rat pituitary. An increase in RNA polymerase I activity, with a maximum effect between 10 and 15 hours, is described. No modification in RNA polymerase II activity was detected. These results were extended and confirmed, using in vitro double labelling of RNA, following in vivo oestrogen treatment. Polyacrylamide gel electrophoresis of nuclear and cytoplasmic RNA showed an increased incorporation of adenine into 28S and 18S rRNA, in the pituitaries of oestrogen-treated animals. The 5S rRNA was not modified by the hormonal treatment. These effects on RNA polymerase I activity and on 28S and 18S rRNA synthesis were closely correlated with the long-term nuclear retention of receptor-oestradiol complexes, in vivo. Taken together, these observations argue in favor of the nucleolus as a preferential target for receptor-bound oestradiol, in the cell nucleus of the male rat pituitary.

Animals↗
PubMed1977

5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei.

A soluble extract prepared from HeLa cell nuclei has been shown to catalyze the 5'-terminal modification of RNA and synthetic polyribonucleotides to form m7G(5')pppA-and m7G(5')-pppG- structures referred to as caps. The reaction involves the transfer of a GMP moiety from GTP to the 5' end of an RNA molecule containing at least two terminal phosphates. Significantly, neither the beta nor the gamma phosphates of GTP are transferred and polynucleotides with no 5'-terminal phosphate or only one are not acceptors. In the absence of methyl donor, G(5')pppA- and G(5')pppG- structures were synthesized, indicating that methylation is not required for guanylylation. Cap formation was considered to occur by the following mechanism: (see article), in which AdoMet is S-adenosylmethionine, AdoHcy is S-adenosylhomocysteine, and (p)ppN- represents either the original 5' end of an RNA molecule or an internal site to which one or more phosphates were added after processing.

Cell Nucleus↗
PubMed1990

Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors.

Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.

Base Sequence↗
PubMed2002

Detection of C8-(1-hydroxyethyl)guanine in liver RNA and DNA from control and ethanol-treated rats.

Alcohol consumption is associated with an increased risk of cancer by mechanisms that remain unknown but have been suggested to involve radical metabolites. We have previously shown that the 1-hydroxyethyl radical produced from ethanol oxidation in vitro is able to alkylate nucleic acids to produce C8-(1-hydroxyethyl)guanine [C8-(1-HE)gua] among other products. To assess if this adduct is produced in vivo, we developed a sensitive HPLC-MS/MS method for its detection and analyzed hydrolysates of liver RNA and DNA from control and ethanol-treated rats. Unexpectedly, C8-(1-HE)gua was found to be present in both RNA and DNA from the liver of control Sprague-Dawley rats, and its levels increased slightly, but not significantly, after an acute ethanol dose (5 g/kg). In rat liver, C8-(1-HE)gua endogenous levels were about 10 times higher in RNA (35 +/- 5/10(7) guanine) than DNA (3.7 +/- 1.1/10(7) guanine). These levels were also found in commercial RNA (calf liver and yeast) and DNA (calf thymus), further indicating the endogenous source of the adduct. DNA basal levels of C8-(1-HE)gua were similar to those reported for other 2C guanine adducts such as N7-(2-hydroxyethyl)guanine and N2-ethyl-2'-deoxyguanosine. We speculate that all of these adducts may be generated from DNA attack by products of basal lipid peroxidation. The higher RNA levels of C8-(1-HE)gua are in agreement with the higher accessibility of RNA and nucleotides to reactive intermediates because they are not as protected or as localized as DNA. Chemical modification of RNA has been receiving increasingly attention as an important event in genotoxic mechanisms. Comparison of RNA basal levels of C8-(1-HE)gua, N7-(2-hydroxyethyl)guanine, and N2-ethyl-2'-deoxyguanosine may provide clues about their endogenous sources and biological significance. Yet, the marginal increase of DNA C8-(1-HE)gua upon ethanol administration argues against this adduct playing a major role in the carcinogenic effects of ethanol.

Animals↗
PubMed2005

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme.

We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this trans excision-splicing ribozyme in a cellular environment, specifically Escherichia coli, was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for trans excision-splicing. We show that the ribozyme is able to catalyze the trans excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.

Escherichia coli↗
PubMed1997

Gender-specific induction of pituitary RNA by estrogen and its modification by thyroid hormone.

Estrogen and thyroid hormones play important roles in the regulation of pituitary function. We presently show that pituitary weight and total cellular RNA levels were significantly decreased by ovariectomy in female rats and were significantly increased by castration in males, without alterations in pituitary DNA levels as compared to intact animals. Treatment with a single dose of estrogen produced a significant increase in pituitary RNA in ovariectomized females but not castrated males. This effect was more obvious following multiple doses of estrogen, and was blocked by pretreatment with cycloheximide, or surprisingly by concomitant administration of triiodothyronine (T3). Analysis of estrogen response element (ERE) binding activity in pituitary nuclear protein extracts revealed that estrogen produced a rapid induction of a slow mobility complex of ERE binding in ovariectomized females much greater than in castrated males. Thus, estrogen-induced increases in pituitary total RNA levels are dependent on new protein synthesis, are gender-specific, are inhibited by T3, and may be mediated via specific estrogen-induced changes in protein-DNA interactions.

Animals↗
PubMed1999

A small catalytic RNA motif with Diels-Alderase activity.

BACKGROUND: The 'RNA world' hypothesis requires that RNA be able to catalyze a wide variety of chemical reactions. In vitro selection from combinatorial RNA libraries has been used to identify several catalytic activities, most of which have resulted in a self-modification of RNA at one of its constituents. The formation of carbon-carbon bonds is considered an essential prerequisite for a complex metabolism based on RNA. RESULTS: We describe the selection and characterization of new ribozymes that catalyze carbon-carbon bond formation by Diels-Alder reaction of a biotinylated maleimide with an RNA-tethered anthracene. Secondary structure analysis identified a 49-nucleotide RNA motif that accelerates the reaction about 20,000-fold. The motif has only 11 conserved nucleotides that are present in most of the selected sequences. The ribozyme motif is remarkably adaptable with respect to cofactor and metal-ion requirements. The motif was also re-engineered to give a 38-mer RNA that can act as a 'true' catalyst on short external substrate oligonucleotide-anthracene conjugates. CONCLUSIONS: We have identified a small, highly abundant RNA motif that can solve the complex task of forming two carbon-carbon bonds between two reactants in trans, a catalytic capacity useful for creating prebiotically relevant molecules. This is the smallest and fastest RNA catalyst for carbon-carbon bond formation reported to date.

Algorithms↗
PubMed1996

An RNA topoisomerase.

A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule. The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments. This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be inter-converted only by a strand passage event. We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase. The conversion of circles to knots is accompanied by a small amount of RNA catenane generation. These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.

Base Sequence↗