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At least 883 records · Page 49Linked to original sources
PubMed1998

Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast.

The messenger RNA 3' poly(A) tail critically affects the initiation and control of translation in eukaryotes. By analogy to elements involved in transcription initiation, the poly(A) tail has been described as a 'translational enhancer' that enhances the 'translational promoter' activity of the mRNA 5'-cap structure. Elongation or shortening of the poly(A) tail regulates translation during development. Here we show, using cell-free and in vivo translation analyses in Saccharomyces cerevisiae, that the poly(A) tail can act as an independent 'translational promoter', delivering ribosomes to uncapped mRNAs even if their 5' end is blocked. When mRNAs compete for ribosome binding, neither the cap structure nor the poly(A) tail alone is enough to drive efficient translation, but together they synergize and direct ribosome entry to the 5' end. The cap structure both promotes ribosome recruitment, together with the poly(A) tail, and tethers recruited ribosomes to the 5' end. Correct choice of translation initiation codons and the function of translational regulators acting on the 5' untranslated region are thus ensured by the functional interaction of the poly(A) tail with the cap structure.

Base Sequence↗
PubMed1998

Subunits of yeast RNA polymerases: structure and function.

Following isolation of the genes encoding the putative subunits of RNA polymerase in both budding and fission yeasts, combined biochemical and genetic studies, together with a structural approach applicable to large assemblies, have begun to reveal the protein-protein interactions not only between RNA polymerase subunits but also between the RNA polymerases and transcription factors. These protein-protein interactions ultimately lead to control of the activity and specificity of the RNA polymerases.

DNA-Directed RNA Polymerases↗
PubMed1998

Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis.

The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic folding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure. We have employed temperature gradient gel electrophoresis (TGGE) to examine the unfolding of the tertiary structure of P4-P6. In 0.9 mM Mg2+, the global tertiary fold of the molecule has a melting temperature of approximately 40 degreesC and is completely unfolded by 60 degreesC. Calculated thermodynamic parameters for folding of P4-P6 are DeltaH degrees' = -28 +/- 3 kcal/mol and DeltaS degrees' = -91 +/- 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfate and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary structure is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetrahymenagroup I intron self-splicing, which have positive or zero DeltaH terms, P4-P6 tertiary structure formation is stabilized by a negative DeltaH term. This implies that enthalpically favorable hydrogen bond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain.

Animals↗
PubMed2003

LNA: a versatile tool for therapeutics and genomics.

Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes ('LNAzyme'). Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.

Biomimetic Materials↗
PubMedDate not supplied

Persistence of nucleolar RNA-rich structures and Ph1 duplication in the blastic crisis of chronic myeloid leukaemia.

Nucleolar persistence in metaphase plates is a feature observed in most of the cells in neoplastic processes. Pathological persistence or fragmentation of the nucleoli is thought to be the cause of some numerical chromosomal aberrations due to non-disjunction of the chromatids, with particular involvement of the satellite chromosomes. Thus, a combined selective staining of both the nucleoli (amido black 10B according to Mundkur and Brauer's cytochemical technique) and the chromosomes (neutral red) was applied to the metaphase plates of patients with chronic myeloid leukaemia in the blastic crisis. Duplicated Ph1 was associated with amido black-stained areas at a very high rate in some cases. Since the blastic crisis in chronic myeloid leukaemia is characterized by the appearance of an increased number of immature, highly nucleolated cells, these findings lend support to the hypothesis that the duplication of the Ph1 represents a feature possibly favoured by the pathological persistence of nucleolar RNA-rich structures in the metaphase.

Adult↗
PubMed1991

Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence.

In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet. Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA. The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes. The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency. On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons. Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs.

Amino Acid Sequence↗
PubMed1989

Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron.

Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E. coli and self-splicing in vitro. Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case. A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels. Thus, we conclude that P8 exists and is essential for splicing. In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8[5'] and P8[3'], results in only a slight decrease in splicing activity. We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron. A two-base mutation in P6[3'] reduces splicing activity to a greater extent than does a two-base mutation in P6[5']. Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing.

Animals↗
PubMed1997

A complex structure in the mRNA of Tf1 is recognized and cleaved to generate the primer of reverse transcription.

All retroviruses and LTR-containing retrotransposons are thought to require specific tRNA molecules to serve as primers of reverse transcription. An exception is the LTR-containing retrotransposon Tf1, isolated from Schizosaccharomyces pombe. Instead of requiring a tRNA, the reverse transcriptase of Tf1 uses the first 11 bases of the Tf1 transcript as the primer for reverse transcription. The primer is generated by a cleavage that occurs between bases 11 and 12 of the Tf1 mRNA. Sequence analysis of the 5' untranslated region of the Tf1 mRNA resulted in the identification of a region with the potential to form an RNA structure of 89 bases that included the primer binding site and the first 11 bases of the Tf1 mRNA. Systematic mutagenesis of this region revealed 34 single-point mutants in the structure that resulted in reduced transposition activity. The defects in transposition correlated with reduced level of Tf1 reverse transcripts as determined by DNA blot analysis. Evidence that the RNA structure did form in vivo included the result that strains with second site mutations that restored complementarity resulted in increased levels of reverse transcripts and Tf1 transposition. The majority of the mutants defective for reverse transcription were unable to cleave the Tf1 mRNA between bases 11 and 12. These data indicate that formation of an extensive RNA structure was required for the cleavage reaction that generated the primer for Tf1 reverse transcription.

Base Composition↗
PubMed1987

Structure of RNA in satellite tobacco necrosis virus. A low resolution neutron diffraction study using 1H2O/2H2O solvent contrast variation.

The crystal structure of satellite tobacco necrosis virus has been studied by neutron diffraction at 16 A resolution using the technique of 1H2O/2H2O solvent contrast variation to distinguish between the regions of protein and nucleic acid. The RNA density is essentially localized in a region just inside the protein coat, leading to a significant interaction between the two components. From the appearance of the RNA density we conclude that the protein coat imposes partial icosahedral symmetry on a significant proportion of the nucleic acid. The shape and dimensions of the major part of this density suggests that about 72% of the total RNA could be double-helical in structure. The most important interaction between the two components of the virus occurs between the N-terminal triple-helical arms of the protein subunits and those regions of the RNA density that could have a double-helical secondary structure.

Amino Acid Sequence↗
PubMed1995

Thermodynamics of RNA unfolding: stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion.

RNAs with interesting secondary and tertiary structures tend to melt in several broad and overlapping transitions over a wide temperature range, and it has been consequently difficult to resolve the thermodynamics of individual unfolding steps. In the case that a ligand selectively binds a single folded state of the RNA, it is possible to obtain reliable thermodynamic parameters for both RNA unfolding and RNA-ligand binding simply from the hyperchromicity of RNA denaturation. The analysis procedure involves fitting a three-dimensional surface to absorbance data collected as a function of both temperature and ligand concentration. Analysis of the unfolding of a fragment of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1109) is presented; both an antibiotic (thiostrepton) and ammonium ion specifically stabilize a tertiary structure within this RNA. A consistent set of thermodynamic parameters (delta H and tm) for the first two sequentially linked unfolding transitions is obtained from the experiments, and the binding constants obtained for the two ligands are consistent with other independent measurements. The approach is applicable to a variety of RNAs that specifically bind proteins, antibiotics, ions or other ligands.

Ammonia↗
PubMed1977

[Ribonucleoproteins containing heterogeneous nuclear and messenger cytoplasmic RNA. Characteristics, structure and relations (author's transl)].

Following the study of Spirin, many authors have shown that cytoplasmic messenger RNA and heterogeneous nuclear RNA are complexed with specific proteins to form ribonucleoprotein particles (RNP). These RNP are heterogeneous in size and present a high protein to RNA ratio. Different observations suggest a polymeric structure for nuclear ribonucleoproteins but their protein composition is more clearly complex than that of cytoplasmic ribonucleoproteins. If we accept the following processing: heterogeneous nuclear RNP leads to free cytoplasmic RNP leads to polysomic RNP, the question arises as to what extent proteins originally present in nuclear RNP are conserved. Proteins more or less tightly bound to RNA have different roles: packing and protection of RNA, regulation during gene expression processing.

Animals↗
PubMed1994

A guanosine quadruplex and two stable hairpins flank a major cleavage site in insulin-like growth factor II mRNA.

Insulin-like growth factor II (IGF-II) mRNAs are cleaved by an endonucleolytic event in a conserved part of their 3' untranslated region that is predicted to exhibit a complex higher-order RNA structure. In the present study, we have examined the putative secondary structures of in vitro transcripts from the conserved part of human and rat mRNAs by enzymatic and chemical probing. The results show that the cleavage site is situated between two highly structured domains. The upstream domain consists of two large hairpins, whereas the downstream domain is guanosine-rich. The guanosine-rich domain adopts a compact unimolecular conformation in Na+ or K+ but not in Li+, and it completely arrests reverse transcription in K+ but only partially in Na+, indicating the presence of an intramolecular guanosine quadruplex. The flanking higher-order structures may ensure that the cleavage site is not sequestered in stable RNA structures, thus allowing interactions with RNA or proteins at posttranscriptional stages of IGF-II expression.

Animals↗
PubMed2002

Quadruplex structures of RNA 14-mer, r(GGAGGUUUUGGAGG) and DNA 14-mer, d(GGAGGTTTTGGAGG).

We have determined solution structure of r (GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. We also synthesized the corresponding DNA 14-mer, d (GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K+ ions; two molecules of D14 form a quadruplex containing antiparalle strands and diagonal loops. The NMR study also gave the results that are consistent with such structure: alternate glycosidic conformation, 5'G(syn)-G(anti)3', and characteristic chemical shift data.

Base Sequence↗
PubMed1991

Genomic structure and RNA polymerase activity in Leishmania virus.

Viral particles infecting some stocks of the protozoan parasite Leishmania braziliensis subsp. guyanensis contain a double-stranded RNA genome of ca. 5 kbp and are associated with an RNA-dependent RNA polymerase which synthesizes in vitro double-stranded and single-stranded, genome-length transcripts. The majority of viral transcripts are single-stranded and templated from one genomic strand. The putative replicase generates double-stranded RNA by synthesizing the opposite strand on a preexisting RNA template. These data are compatible with a replicative cycle proposed for the yeast viruses. Purification of the Leishmania virus on CsCl yields virus without double-strand synthesis activity, while this activity is consistently present in unpurified virus and in particles from sucrose gradients. The deficiency in double-strand synthesis in CsCl-derived virions correlates with the accessibility of the viral polymerase and genomic RNA to exogenously added enzymes, indicative of a structural modification of the viral capsid.

Animals↗
PubMed2005

Structural perspective on the activation of RNAse P RNA by protein.

Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

Bacteria↗
PubMed1994

Mapping tRNA and 5S RNA tertiary structures by charge dependent Fe(II)-catalyzed cleavage.

Chemical and enzymatic footprinting experiments have made it possible to identify protein binding sites in DNA and RNA, and to localize structural differences within nucleic acids to a resolution of a single base pair. We show here that by combining three reagents, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, differential maps of sites in RNA that vary in their local conformation and/or charge can be constructed. Comparison of profiles with respect to controls in the absence of a counterion such as Mg2+ allows analysis of sites responsive to tertiary structure. A single site that is labile to metals such as Pb2+ exists in tRNA(Phe) and a number of other tRNA's; this site is hyper-reactive to Fe(II), but not to the other probes. Scission induced by the neutral complex, Fe(II).EDDA, offers the most general measure of surface accessibility, since its distribution about the target molecule is insensitive to charge. Enhanced cleavage by Fe(II) relative to the other agents is detected at several adjacent sites in 5S RNA, consistent with conformational mobility. Protection at a series of positions in the arm formed by loops E and D with helix IV suggests further that at low temperature this arm interacts with loop A and helix I.

Base Composition↗