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At least 721 records · Page 40Linked to original sources
PubMed2003

Synergism between p68 RNA helicase and the transcriptional coactivators CBP and p300.

p68 RNA helicase has been implicated in a variety of processes, including rearrangement of RNA secondary structures, RNA splicing, gene transcription and tumor development, yet its mechanisms of action are not well understood. In this study, we show that p68 is predominantly localized to the cell nucleus, where it partially colocalizes with the transcriptional coactivator p300. Accordingly, p68 and p300, or the paralogous CREB-binding protein (CBP), coimmunoprecipitate. Similarly, p68 and RNA polymerase II (Pol II) are able to interact in vivo. GST pull-down assays confirmed these interactions in vitro, demonstrating that p68 can interact with several domains of CBP, while CBP/p300 bind to amino acids 176-388 of p68 and RNA Pol II binds to the N-terminal 80 amino acids of p68. Furthermore, p68 stimulates transcription mediated by the C-terminal transactivation domain of CBP. p68 is also able to stimulate TPA oncogene responsive unit (TORU) promoter activity, and p300 acts in synergy with p68. On the other hand, suppression of CBP/p300 function by the adenoviral protein E1A abolishes TORU promoter activation by p68. Altogether, our results suggest the existence of a multiprotein complex in which p68 RNA helicase, CBP/p300 and RNA Pol II jointly promote gene expression.

Cell Line↗
PubMed1971

Prediction of RNA secondary structure.

Calculations of the free energy required to close single-strand loops by formation of a base pair in double-helical nucleic acids are reported. These results can be used to estimate the free energy of particular secondary structures for a given RNA molecule under conditions of high-salt concentration.

Models, Theoretical↗
PubMed2000

Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals.

Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.

DNA-Directed RNA Polymerases↗
PubMed1968

Effect of fluoride on nucleotides and ribonucleic Acid in germinating corn seedling roots.

Investigation was made for the effect of fluoride on plant growth, acid soluble nucleotides, and RNA in germinating corn seedling roots. Fluoride suppresses root growth as measured by changes in fresh weight. Column chromatographic analyses demonstrated that fluoride modifies ratios of acid soluble nucleotide species. The relative amount of nucleotides is altered mainly due to triphosphate nucleotides of which ATP is most accumulated. Paper chromatographic analyses showed that fluoride induces changes of RNA structure. The RNA is characterized by lowered relative content of cytosine and by increased ratio of cytosine to guanine. Adenine is depressed significantly only in the root tissue treated by the highest fluoride concentration.

Journal Article↗
PubMed1970

Cytoplasmic extraction: polyribosomes and heterogenous ribonucleoproteins without associated DNA.

Two methods are described for preparing cytoplasmic extracts from sea urchin embryos. One method, involving homogenization, yields DNA structures that cosediment with polyribosomes and subribosomal ribonucleoproteins. In addition this method also yields extraneous structures containing RNA that cosediment with polyribosomes. The DNA is not associated with polyribosomes, as shown by buoyant density analysis. Furthermore, this DNA appears to be spurious, because its release into a cytoplasmic extract does not occur when a different method of cell disruption, involving passage of embryos through a hypodermic needle, is used. With this second method, polyribosomes are obtained without extraneous cosedimenting RNA structures and subribosomal ribonucleo-proteins are obtained in the virtual absence of DNA.

Animals↗
PubMed1990

bicoid mRNA localization signal: phylogenetic conservation of function and RNA secondary structure.

Transcripts of the bicoid (bcd) gene are localized to the anterior pole of the Drosophila oocyte, thereby allowing formation in the embryo of an anteroposterior gradient of the bcd protein morphogen. We previously showed that a 630 nucleotide portion of the 3' noncoding region of the bcd mRNA is necessary for this localization, and is sufficient to confer anterior localization on a heterologous transcript. Here I have used a comparative analysis to begin to more precisely define the cis-acting mRNA localization signal. The bcd genes from six additional Drosophila species were cloned, and DNA of the 3' noncoding regions sequenced. Three of these regions were tested interspecifically for mRNA localization in D. melanogaster and each functioned correctly; these regions must therefore contain the cis-acting signal. The primary sequences, which are up to 50% divergent from the D. melanogaster gene, show patchy homology throughout most of the region. Interestingly, all seven species can potentially form a large stereotypic secondary structure. This structure is a likely candidate for the localization signal and can be used for the rational design of mutations to test that possibility.

Animals↗
PubMed1994

The theory of genotypic selection: predicting the direction of evolution as a consequence of G:U base pairing and the existence of non-pathogenic strains of HIV-1.

One aspect of the Theory of Genotypic Selection states that G:U base pairs are selected for function and structure. RNA secondary structures stabilised by G:U base pairs are suggested to be involved in the evolution of attenuated strains of polio virus. Asymmetric mutations (C-->U and A-->G) and convergent evolutionary solutions are explained as a direct consequence of G:U base pairing. The possibility of a naturally occurring attenuated strain of HIV-1 is predicted.

Base Composition↗
PubMed1995

Mitochondrial transcription initiation: promoter structures and RNA polymerases.

A diversity of promoter structures. It is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures. Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional. In fungi and plants, multiple promoters are found, and in each case, both the extent and the primary sequences of promoters are distinct. Promoter multiplicity in fungi, plants and trypanosomes reflects the larger genome size and scattering of genes relative to animals. However, the dual roles of certain promoters in transcription and replication, at least in yeast, raises the interesting question of how the relative amounts of RNA versus DNA synthesis are regulated, possibly via cis-elements downstream from the promoters. Mitochondrial RNA polymerases. With respect to mitochondrial RNA polymerases, characterization of human, mouse, Xenopus and yeast enzymes suggests a marked degree of conservation in their behavior and protein composition. In general, these systems consist of a relatively non-selective core enzyme, which itself is unable to recognize promoters, and at least one dissociable specificity factor, which confers selectivity to the core subunit. In most of these systems, components of the RNA polymerase have been shown to induce a conformational change in their respective promoters and have also been assigned the role of a primase in the replication of mtDNA. While studies of the yeast RNA polymerase have suggested it has both eubacterial (mtTFB) and bacteriophage (RPO41) origins, it is not yet clear whether these characteristics will be conserved in the mitochondrial RNA polymerases of all eukaryotes. mtTFA-mtTFB; conserved but dissimilar functions. With respect to transcription factors, mtTFA has been found in both vertebrates and yeast, and may be a ubiquitous protein in mitochondria. However, the divergence in non-HMG portions of the proteins, combined with differences in promoter structure, has apparently relegated mtTFA to alternative, or at least non-identical, physiological roles in vertebrates and fungi. The relative ease with which mtTFA can be purified (Fisher et al. 1991) suggests that, where present, it should be facile to detect. mtTFB may represent a eubacterial sigma factor adapted for interaction with the mitochondrial RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

An RNA secondary structure juxtaposes two remote genetic signals for human T-cell leukemia virus type I RNA 3'-end processing.

Sequence analysis of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) does not reveal a polyadenylation consensus sequence, AAUAAA, close to the polyadenylation site at the 3' end of the viral RNA. Using site-directed mutagenesis, we demonstrated that two cis-acting signals are required for efficient RNA processing in HTLV-I LTR: (i) a remote AAUAAA hexamer at a distance of 276 nucleotides upstream of the polyadenylation site, and (ii) the 20-nucleotide GU-rich sequence immediately downstream from the poly(A) site. It has been postulated that the folding of RNA into a secondary structure juxtaposes the AAUAAA sequence, in a noncontiguous manner, to within 14 nucleotides of the polyadenylation site. To test this hypothesis, we introduced deletions and point mutations within the U3 and R regions of the LTR. RNA 3'-end processing occurred efficiently at the authentic HTLV-I poly(A) site after deletion of the sequences predicted to form the secondary structure. Thus, the genetic analysis supports the hypothesis that folding of the HTLV-I RNA in the U3 and R regions juxtaposes the AAUAAA sequence and the poly(A) site to the correct functional distance. This unique arrangement of RNA-processing signals is also found in the related retroviruses HTLV-II and bovine leukemia virus.

Base Sequence↗
PubMed2005

Moments of the Boltzmann distribution for RNA secondary structures.

We here present a dynamic programming algorithm which is capable of calculating arbitrary moments of the Boltzmann distribution for RNA secondary structures. We have implemented the algorithm in a program called RNA-variance and investigate the difference between the Boltzmann distribution of biological and random RNA sequences. We find that the minimum free energy structure of biological sequences has a higher probability in the Boltzmann distribution than random sequences. Moreover, we show that the free energies of biological sequences have a smaller variance than random sequences and that the minimum free energy of biological sequences is closer to the expected free energy of the rest of the structures than that of random sequences. These results suggest that biologically functional RNA sequences not only require a thermodynamically stable minimum free energy structure, but also an ensemble of structures whose free energies are close to the minimum free energy.

Algorithms↗
PubMed2004

A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing.

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a CFTR minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen in vivo. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.

Base Sequence↗
PubMed2004

MSARI: multiple sequence alignments for statistical detection of RNA secondary structure.

We present a highly accurate method for identifying genes with conserved RNA secondary structure by searching multiple sequence alignments of a large set of candidate orthologs for correlated arrangements of reverse-complementary regions. This approach is growing increasingly feasible as the genomes of ever more organisms are sequenced. A program called msari implements this method and is significantly more accurate than existing methods in the context of automatically generated alignments, making it particularly applicable to high-throughput scans. In our tests, it discerned clustalw-generated multiple sequence alignments of signal recognition particle or RNaseP orthologs from controls with 89.1% sensitivity at 97.5% specificity and with 74.4% sensitivity with no false positives in 494 controls. We used msari to conduct a comprehensive scan for secondary structure in mRNAs of coding genes, and we found many genes with known mRNA secondary structure and compelling evidence for secondary structure in other genes. msari uses a method for coping with sequence redundancy that is likely to have applications in a large set of other comparison-based search methods. The program is available for download from http://theory.csail.mit.edu/MSARi.

Algorithms↗
PubMed1993

Prediction of alternative RNA secondary structures based on fluctuating thermodynamic parameters.

In this paper we present a new method for predicting a set of RNA secondary structures that are thermodynamically favored in RNA folding simulations. This method uses a large number of 'simulated energy rules' (SER) generated by perturbing the free energy parameters derived experimentally within the range of the experimental errors. The structure with the lowest free energy is computed for each SER. Structural comparisons are used to avoid multiple generation of similar structures. Computed structures are evaluated using the energy distribution of the lowest free energy structures derived in the simulation. Predicted be graphically displayed with their occurring frequencies in the simulation by dot-plot representations. On average, about 90% of phylogenetic helixes in the known models of tRNA, Group I self-splicing intron, and Escherichia coli 16 S rRNA, were predicted using the method.

Algorithms↗
PubMed1978

Analysis of terminal structures of RNA from potato virus X.

The 5'-end structure of potato virus X RNA was determined following enzymatic methylation in vitro. A single 3H-methyl group was introduced into the 2'-position of the 5'-penultimate residue and the end structure was determined as m7GpppG(m)pAp(Xp)3G. This part of the RNA apparently is involved in binding to ribosomes since it can be partially protected against RNase digestion by wheat germ 40S ribosomes. PVX RNA was not retained by poly(U)-sepharose, indicating that it does not contain a 3'-terminal poly(A) tract.

Base Sequence↗
PubMed2005

A class of 2D graphical representations of RNA secondary structures and the analysis of similarity based on them.

Based on the concepts of cell and system of graphical representation, a class of 2D graphical representations of RNA secondary structures are given in terms of classifications of bases of nucleic acids. The representations can completely avoid loss of information associated with crossing and overlapping of the corresponding curve. As an application, we make quantitative comparisons for a set of RNA secondary structures at the 3'-terminus of different viruses based on the graphical representations. The examination of similarities/dissimilarities illustrates the utility of the approach.

Algorithms↗
PubMed2003

Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis.

The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

Base Sequence↗
PubMed1991

Structural basis for the regulation of splicing of a yeast messenger RNA.

In S. cerevisiae, ribosomal protein L32 regulates the splicing of the transcript of its own gene, RPL32. We have identified an RNA structure within the transcript that is responsible for this regulation. Initial deletions limited essential sequences to the 5' exon and the first few nucleotides of the intron. To take advantage of phylogenetic comparison of RNA structures, RPL32 was cloned from the closely related species, Kluyveromyces lactis. The splicing of its transcript is similarly regulated. Sequences conserved between the S. cerevisiae and K. lactis transcripts suggested a structure involving base pairing of a region encompassing the 5' splice site with another near the 5' end of the transcript. Analysis of numerous site-directed mutations supports this structure. We infer that stabilization of this structure by L32 inhibits splicing by precluding the interaction of U1 RNA with the 5' splice site.

Base Sequence↗
PubMed2004

Modified bases in RNA reduce secondary structure and enhance hybridization.

Secondary structure in RNA targets is a significant barrier to short DNA probes. However, when such targets are the end product of an in vitro amplification scheme, it is possible to carry out transcription in the presence of nucleoside triphosphate analogues that reduce secondary structure of the RNA without impairing subsequent hybridization. Here we show that nucleoside triphosphates of 2-aminoadenine (nA) and 2-thiouracil (sU) are taken up by T7 RNA polymerase and that the resulting RNA possesses reduced secondary structure and improved accessibility to DNA probes. The hybridization properties of short RNA transcripts were studied using a new gel mobility shift assay from which melting temperatures were determined. RNA hairpins that contained nA and sU were able to hybridize to DNA probes under conditions where the unmodified hairpins did not. DNA-RNA hybrids that contained nA and sU in the RNA strand exhibited enhanced specificity, increased stability, and greater equality of base pairing strength than the same hybrids without modifications. Substitution of guanine (G) with inosine (I) further reduced secondary structure, but RNA with this base hybridized nonselectively. The high stability of nA-T and A-sU base pairs in DNA-RNA hybrids, combined with the destabilizing effect of the nA-sU couple in RNA targets, accounts for the improved hybridization properties. These results suggest that incorporation of nA and sU during in vitro transcription is a promising strategy for enhancing the performance of oligomeric DNA probes with an RNA target.

2-Aminopurine↗