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At least 757 records · Page 42Linked to original sources
PubMed2005

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines.

Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4'-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4'-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s2U) and 5-methyl-2-thiouridine (m5s2U) are natural nucleotide analogs. s2U in tRNA confers greater specificity of codon-anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2'-deoxyribose sugars for a C3'-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2'-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2'-O-[2-(methoxy)ethyl]-2-thiothymidine (m5s2Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m5s2Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m5Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter 'opening', an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m5s2Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2'-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.

Base Pairing↗
PubMed1997

The primer binding site on the RNA genome of human and simian immunodeficiency viruses is flanked by an upstream hairpin structure.

Reverse transcription of retroviral genomes is primed by a tRNA molecule that anneals to an 18 nt primer binding site (PBS) on the viral RNA genome. Additional base pair interactions between the tRNA primer and the viral RNA have been proposed. In particular, base pairing was proposed between the anticodon loop of tRNALys3 and the 'A-rich' loop of a hairpin located immediately upstream of the PBS site in HIV-1 RNA. In order to judge the importance of this sequence/structure motif, we performed an extensive phylogenetic analysis of this genomic region in a variety of simian and human immunodeficiency viruses (SIV and HIV). Both the phylogeny of natural HIV/SIV sequences and the behaviour of U5-PBS mutant/revertant viruses support the idea that this RNA structure is critical for virus replication. Although this hairpin may play a role in tRNA annealing and/or initiation of reverse transcription, the proposed base pairing interaction between the A-rich loop of the HIV-1 hairpin and the anticodon of the initiator tRNA is not directly supported by this analysis.

Base Composition↗
PubMed1992

Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group.

In order to elucidate roles of the 2'-O-methylation of pyrimidine nucleotide residues of tRNAs, conformations of 2'-O-methyluridylyl(3'----5')uridine (UmpU), 2'-O-methyluridine 3'-monophosphate (Ump), and 2'-O-methyluridine (Um) in 2H2O solution were analyzed by one- and two-dimensional proton NMR spectroscopy and compared with those of related nucleotides and nucleoside. As for UpU and UmpU, the 2'-O-methylation was found to stabilize the C3'-endo form of the 3'-nucleotidyl unit (Up-/Ump-moiety). This stabilization of the C3'-endo form is primarily due to an intraresidue effect, since the conformation of the 5'-nucleotidyl unit (-pU moiety) was only slightly affected by the 2'-O-methylation of the 3'-nucleotide unit. In fact even for Up and Ump, the 2'-O-methylation significantly stabilizes the C3'-endo form by 0.8 kcal/.mol-1. By contrast, for nucleosides (U and Um), the C3'-endo form is slightly stabilized by 0.1 kcal/.mol-1. Accordingly, the stabilization of the C3'-endo form by the 2'-O-methylation is primarily due to the steric repulsion among the 2-carbonyl group, the 2'-O-methyl group and the 3'-phosphate group in the C2'-endo form. For some tRNA species, 2-thiolation of pyrimidine residues is found in positions where the 2'-O-methylation is found for other tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)

Dinucleoside Phosphates↗
PubMed1983

Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids.

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Sucrose modifies ribosomal stability and conformation.

High concentrations of sucrose have a strong protective effect on heat-induced modifications of rat liver ribosomal subunits. They prevent to a large extent subunit inactivation, measured by poly (U)-dependent [14C] Phe tRNA binding (40S subunits) and puromycin reaction (60S subunits), subunit unfolding into light forms, and the release of both free and protein-complexed 5S RNA. They also increase the temperature at which subunits start to melt. Our data indicate that sucrose affects subunit conformation.

Animals↗
PubMed1993

Characterization of aminoacyl-adenylates in B. subtilis tryptophanyl-tRNA synthetase, by the fluorescence of tryptophan analogs 5-hydroxytryptophan and 7-azatryptophan.

The tryptophan analogs 5-hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are capable of being biosynthetically incorporated into bacterial proteins and can be used as intrinsic fluorescence probes of protein structure, function and dynamics. A prerequisite for analog incorporation is their recognition by tryptophanyl-tRNA synthetase (TrpRS) and the formation of the analog aminoacyladenylate in the enzyme's active site. The binding of 5HW and 7AW to B. subtilis TrpRS and the stability of the corresponding aminoacyladenylates of 5HW and 7AW were examined using their unique spectroscopic properties. The adenylate of 7AW in the active site of TrpRS exhibited intense fluorescence with a 10.5 ns fluorescence decay time. Enzyme-bound 7AW-adenylate was a long-lived intermediate with a half-life of over 9 hours. Enzyme-bound 5HW-adenylate fluorescence was quenched compared to that of 5HW in solution. The 5HW-adenylate/TrpRS complex was much less stable than that of 7AW, with a half-life of 33 minutes. Rapid hydrolysis of the 5HW-adenylate may explain the apparent proofreading observed which prohibits 5HW incorporation into proteins in the presence of tryptophan. Hydrolysis of the adenylates of both analogs restored the fluorescence parameters towards those of the analogs in solution. Neither 1-methyltryptophan nor 5-methoxytryptophan were capable of forming long-lived aminoacyladenylate intermediates in TrpRS. This study provides perspectives on the usefulness of 5HW and 7AW as intrinsic fluorescence probes of protein structure. The enhanced fluorescence of 7AW suggests its location in a buried hydrophobic environment in the protein. Exposure to water results in significant fluorescence quenching. These studies clearly demonstrate the utility of Trp analogs for the elucidation of molecular details of protein structure and dynamics.

5-Hydroxytryptophan↗
PubMed1995

The binding of apobec-1 to mammalian apo B RNA is stabilized by the presence of complementation factors which are required for post-transcriptional editing.

C to U RNA editing in mammalian intestinal apolipoprotein B mRNA creates an in-frame translational stop and the synthesis of a truncated protein called apo B48. This site specific cytidine deamination is mediated by an enzyme complex of which the catalytic component (apobec-1) is a 27 kDa zinc-binding protein. apobec-1, expressed in bacteria, will bind to mammalian apo B RNA as well as a number of other AU-rich RNA templates. Apo B RNA-binding activity can be competed by the addition of tRNA, an effect which can be overcome by the addition of complementation factors such as chick enterocyte S-100 extracts. Thus, apobec-1 may be a non-specific RNA binding protein which requires the presence of complementation factors to stabilize and enhance its binding in the setting of the holo-enzyme.

APOBEC-1 Deaminase↗
PubMed1983

Analysis of ribosomal RNA in human fibroblasts continuously treated with hydrocortisone.

The prolongation of the limited lifespan of human embryonic lung fibroblasts by hydrocortisone was previously associated with increased uridine incorporation into ribosomal RNA (rRNA). Using polyacrylamide gel electrophoresis, we show here that the continuous treatment with 12 microM hydrocortisone results in increased uridine incorporation into rRNA precursors in cells stimulated to grow by serum, but not in resting cells, suggesting that the hormone does not directly activate rRNA transcription. Chase experiments indicate that the stability of 28S and 18S rRNA is enhanced by hydrocortisone in quiescent cells but not after serum refeeding. Similar results were also observed for transfer RNA (tRNA). Therefore, these effects of hydrocortisone seem to be indirect consequences of pleiotropic actions of glucocorticoid, such as stimulation of processes involved in cell proliferation and inhibition of lysosomal enzymes.

Blood↗
PubMed1983

Molecular dynamics of phenylalanine transfer RNA.

The atomic motions of yeast phenylalanine transfer RNA have been simulated using the molecular dynamics algorithm. Two simulations were carried out for a period of 12 picoseconds, one with a normal Van der Waals potential and the other with a modified Van der Waals potential intended to mimic the effect of solvent. An analysis of large scale motions, surface exposure, root mean square displacements, helical oscillations and relaxation mechanisms reveals the maintenance of stability in the simulated structures and the general similarity of the various dynamic features of the two simulations. The regions of conformational flexibility and rigidity for tRNA(Phe) have been shown in a quantitative measure through this approach.

Computer Simulation↗
PubMed2002

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation.

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.

Eukaryotic Initiation Factor-2↗
PubMed2005

Cell cycle-regulated degradation of tmRNA is controlled by RNase R and SmpB.

The production and removal of regulatory RNAs must be controlled to ensure proper physiological responses. SsrA RNA (tmRNA), a regulatory RNA conserved in all bacteria, is cell cycle regulated and is important for control of cell cycle progression in Caulobacter crescentus. We report that RNase R, a highly conserved 3' to 5' exoribonuclease, is required for the selective degradation of SsrA RNA in stalked cells. Purified RNase R degrades SsrA RNA in vitro, and is kinetically competent to account for all SsrA RNA turnover. SmpB, a tmRNA-binding protein, protects SsrA RNA from RNase R degradation in vitro, and the levels of SmpB protein during the cell cycle correlate with SsrA RNA stability. These results suggest that SmpB binding controls the timing of SsrA RNA degradation by RNase R. We propose a model for the regulated degradation of SsrA RNA in which RNase R degrades SsrA RNA from a non-tRNA-like 3' end, and SmpB specifically protects SsrA RNA from RNase R. This model explains the regulation of SsrA RNA in other bacteria, and suggests that a highly conserved regulatory mechanism controls SsrA activity.

Caulobacter crescentus↗
PubMed1989

Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

Adenosine↗