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At least 1,513 records · Page 84Linked to original sources
PubMed1976

Plant Desiccation and Protein Synthesis: III. Stability of Cytoplasmic RNA during Dehydration, and Its Synthesis on Rehydration of the Moss Tortula ruralis.

RNA species from the haploid gametophyte generation of the moss Tortula ruralis exhibit typical eukaryotic characteristics. The major ribosomal and soluble RNA species are stable during drying and rehydration. RNA synthesis occurs rapidly on reintroduction of the moss to water and incorporation into high molecular weight RNA fractions was detected after 20 to 30 minutes of rehydration and into low molecular weight fractions after 30-60 minutes. Newly synthesized ribosomal RNA was detected in ribosomes within 2 hours of rehydration, but not in polysomes. It is apparent that the ribosomal and transfer RNA conserved during desiccation is involved in the re-establishment of early protein synthesis during subsequent rehydration and that, initially, there is no requirement for newly synthesized material.

Journal Article↗
PubMed1965

Hormone-induced stabilization of soluble RNA in pea-stem tissue.

When indoleacetic acid labeled with carbon-14 in the carboxyl group is fed to excised green pea-stem segments, growth is initiated, and there is a parallel progressive labeling of the RNA extracted by cold phenol. The bulk of the label is found in the 4S fraction. This fraction is more resistant to degradation by ribonuclease than a similar fractian obtained from tissue not treated with indoleacetic acid.

Carbon Isotopes↗
PubMed1992

Stability of Drosophila RNA polymerase II elongation complexes in vitro.

We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.

Actins↗
PubMed2006

The SL1-SL2 (stem-loop) domain is the primary determinant for stability of the gamma retroviral genomic RNA dimer.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

Base Sequence↗
PubMed1996

The Oct-1 POU-specific domain can stimulate small nuclear RNA gene transcription by stabilizing the basal transcription complex SNAPc.

The RNA polymerase II and III human small nuclear RNA promoters have a common basal element, the proximal sequence element, which binds the TATA box-binding protein-containing complex SNAPc. They also contain an enhancer characterized by a highly conserved octamer sequence, which constitutes a binding site for the broadly expressed POU domain transcription factor Oct-1. The POU domain is a bipartite DNA-binding domain consisting of a POU-homeo (POUH) domain and a POU-specific (POUs) domain joined by a flexible linker. Here, we show that the Oct-1 POU domain but not the related Pit-1 POU domain can facilitate the binding of SNAPc to the proximal sequence element, and activate transcription. The effect is probably mediated by protein-protein contacts, and 1 of 30 amino acid differences between the Oct-1 and Pit-1 POUs domains is the key determinant for the differential interaction with SNAPc and the ability to activate transcription. These results show that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain. In this case, subtle changes between activator DNA-binding domains, as subtle as a single amino acid difference, can profoundly affect interaction with the basal transcription machinery.

Amino Acid Sequence↗
PubMed2005

Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Actins↗
PubMed1975

Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA.

Pairs of very closely related Escherichia coli strains were prepared, one having the wild-type allele for ribonuclease III, an enzyme which specifically degrades double-stranded RNA, and the other having a mutant RNase III allele. Growth and phage plating efficiency were compared in these strains. The RNase III+ strains grow better than the RNase III- strains and plate T7 and lambda phage better, but T4 plates with the same efficiency on both strains. On the other hand, the half lives of newly synthesized RNA as well as of functional beta-galactosidase mRNA are similar in both kind of strains. These two parameters, however, are significantly longer in both strains as compared to the original strain from which they were derived. Also, no difference in the differential induction of beta-galactosidase was observed between such strains. Thus, we have to conclude that either ribonuclease III does not play a significant role in the functioning and stability of newly synthesized mRNA, or that enough enzymatic activity was left, residual RNase III or some other enzyme to deal with double-stranded regions in the message.

Enzyme Induction↗
PubMed1991

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

Animals↗
PubMed2003

Design of molecular beacons for AmpliDet RNA assay--characterization of binding stability and probe specificity.

AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe-target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these deltaG(0) parameters, which was compared to experimental analysis. The average absolute discrepancies for deltaG(0)(41) and for the melting temperatures of MB secondary structures were 0.30 +/- 0.26 kcal/mol and 2.15 +/- 1.5 degrees C, respectively. deltaG(0)(41) of probe-target interaction was predicted with a discrepancy of 1.2 +/- 1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.

Base Sequence↗
PubMed2001

Elements of thermodynamics in RNA evolution.

The paper presents some aspects correlating thermal stability of RNA folding and the occurrence of structural motifs in natural ribonucleic acids. Particularly, the thermodynamic stability of 2'-5' and 3'-5' linked RNA and the contribution of unpaired terminal nucleotides (dangling ends) in secondary (2D) and tertiary (3D) structures of RNA are discussed. Both examples suggest that during evolution nature selected sequences and structures of RNA which are the most thermally stable and efficient for their biological function.

Base Sequence↗
PubMed2005

The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase.

The relative steady state concentration of mRNAs of four housekeeping single-copy type Trypanosoma cruzi genes (actin, triosephosphate isomerase, trypanothion reductase and the ribosomal protein S4) was analyzed throughout the growth curve. A distinguishable pattern was observed with maximal levels occurring at the logarithmic phase of growth and minimum levels occurring at the stationary phase. The half-lives of all analyzed messenger RNAs, and also of three molecular species of immature ribosomal RNAs were increased in cells isolated from stationary phase. These results suggest the occurrence of a novel global regulation mechanism that might protect transcripts from degradation in stationary epimastigotes, probably as a strategy to perpetuate through this quiescent stage.

Animals↗
PubMed1995

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.

Base Sequence↗
PubMed2006

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo.

RNA loop-loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop-loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative beta-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5' A(A)/(G)N6A 3'), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.

Base Pairing↗