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At least 1,711 records · Page 95Linked to original sources
PubMed2000

Specific binding of Hoechst 33258 to site 1 thymidylate synthase mRNA.

The translational initiator codon in thymidylate synthetase (TS) mRNA is located in a stem-loop structure with a CC bubble. TS is an important target for anticancer drugs. Aminoglycoside antibiotics have been shown to specifically bind to TS mRNA site 1 constructs and, furthermore, specific binding requires the non-duplex CC bubble region. It is shown here that DNA intercalating agents and DNA minor groove-binding drugs also bind to a TS mRNA site 1 construct. This binding is competitive with aminoglycosides, suggesting that the binding sites overlap. Hoechst 33258 binds with a dissociation constant of 60 nM, a value significantly lower than the approximately 1 microM values found for aminoglycosides. Footprinting and direct binding studies show that the CC bubble is important for binding of the Hoechst compound. However, the exact structure of the bubble is unimportant. Interestingly, mutations in regions adjacent to the bulge also affect binding. These studies point to the important role of non-duplex RNA structures in binding of the DNA minor groove binder Hoechst 33258.

Anti-Bacterial Agents↗
PubMed1983

Comparative structural analysis of cytoplasmic and chloroplastic 5S rRNA from spinach.

5S rRNAs from Spinacea oleracea cytoplasmic and chloroplastic ribosomes have been subjected to digestion with the single strand specific nuclease S1 and to chemical modification of cytidines by sodium bisulphite in order to probe the RNA structure. According to these data, cytoplasmic 5S rRNA can be folded as proposed in the general eukaryotic 5S rRNA structure (1) and 5S rRNA from chloroplastides is shown to be more related to the general eubacterial structure (2).

Base Sequence↗
PubMed1983

Structure of the ribosome-associated 5.8 S ribosomal RNA.

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.

Animals↗
PubMed2003

Ribozymes: recent advances in the development of RNA tools.

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.

Animals↗
PubMed1994

Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA.

Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.

Journal Article↗
PubMed1997

In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.

Cold Temperature↗
PubMed1997

The crystal structure of an RNA oligomer incorporating tandem adenosine-inosine mismatches.

The X-ray crystallographic structure of the RNA duplex [r(CGCAIGCG)]2 has been refined to 2.5 A. It shows a symmetric internal loop of two non-Watson-Crick base pairs which form in the middle of the duplex. The tandem A-I/I-A pairs are related by a crystallographic two-fold axis. Both A(anti)-I(anti) mismatches are in a head-to-head conformation forming hydrogen bonds using the Watson-Crick positions. The octamer duplexes stack above one another in the cell forming a pseudo-infinite helix throughout the crystal. A hydrated calcium ion bridges between the 3'-terminal of one molecule and the backbone of another. The tandem A-I mismatches are incorporated with only minor distortion to the backbone. This is in contrast to the large helical perturbations often produced by sheared G-A pairs in RNA oligonucleotides.

Adenosine↗
PubMed2005

RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction.

The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.

Bacterial Proteins↗
PubMed1988

High resolution structure of the RNA duplex [U(U-A)6A]2.

RNA is involved in many biological functions, ranging from information storage and transfer to the catalysis of reactions involving both nucleic acids and proteins. Previous crystallographic studies on RNA oligomeric chains provide only averaged structures or information limited in resolution. The oligomer [U(U-A)6A]2 was chosen for the study of protein-RNA interactions in viruses. Its size and base composition mimic portions of the genomic RNA in alfalfa mosaic virus that bind to the amino terminus of the viral subunit. The actual sequence was designed to guarantee the formation of a single species of duplex and to facilitate the production of the pure oligomer in large quantities. The molecular structure, derived from the 2.25 A resolution X-ray diffraction data, allows the most detailed analysis of an A-RNA helix reported to date. Two kinks are observed that divide the duplex into three blocks, each close to a canonical A-helical conformation. A few intermolecular hydrogen bonds involving 2'-hydroxyl groups stabilize this peculiar conformation of the RNA, which may be related to the temperature used for the crystallization (35 degrees C). The structure demonstrates both the plasticity of the RNA molecule and the role of the 2'-hydroxyl groups in intermolecular interactions.

Base Sequence↗
PubMed2000

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1.

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.

Binding Sites↗
PubMed2006

Expression of CD83 is regulated by HuR via a novel cis-active coding region RNA element.

Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.

Active Transport, Cell Nucleus↗
PubMed1999

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.

Animals↗
PubMed2005

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex.

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.

Binding Sites↗
PubMed1995

Frequent use of the same tertiary motif by self-folding RNAs.

We have identified an 11 nucleotide RNA motif, [CCUAAG...UAUGG], that is extraordinarily abundant in group I and group II self-splicing introns at sites known, or suspected from co-variation analysis, to interact with hairpin terminal loops with a GNRA consensus sequence. Base substitution experiments using a ribozyme-substrate system derived from a group I intron reveal that this motif interacts preferentially with GAAA terminal loops and binds them with remarkable affinity, compared with other known combinations of GNRA loops and matched targets. A copy of the [CCUAAG...UAUGG] motif which is present in domain I of many group II introns is shown to interact with the GAAA terminal loop that caps domain V. This is the first interaction to be identified between these two domains, whose mutual recognition is known to be necessary and sufficient for group II ribozymic activity. We conclude that interaction of [CCUAAG...UAUGG] with GAAA loops is one of the most common solutions used by nature to solve the problem of compacting and bringing together RNA structural domains.

Base Sequence↗
PubMed1994

Nucleic acid structure analysis. Mathematics for local Cartesian and helical structure parameters that are truly comparable between structures.

Analyzing nucleic acid structures in a comparable manner has become increasingly important as the number of solved structures has increased. This paper presents the concepts, mathematics, theorems, and proofs that form the basis of a new program to analyze three-dimensional DNA and RNA structures. The approach taken here provides numerical data in accordance with guidelines set at a 1988 EMBO workshop. Mathematical definitions are provided for all local structural parameters described in the guidelines. The definitions satisfy the guideline requirements while preserving the original physical intuition of the parameters. In particular, the rotational parameters are true rotations based on a simple physical model (net rotation at constant angular velocity), not Euler angles or angles between vectors and planes as is the case with other approaches. As a result, the mathematical definitions are symmetrical with the property that a 5 degrees tilt is the same as a 5 degrees roll and a 5 degrees twist, except that the rotations take place about different axes. In other approaches, a 5 degrees tilt can mean a different amount of net rotation than a 5 degrees roll or a 5 degrees twist. A second unique feature of the mathematics is that it explicitly incorporates the concept of a pivot point, which is the point about which a base in a base-pair rotates as it buckles, propeller twists, and opens. Pivot points enable one to model the physical motion of bases more accurately. As a result, they greatly reduce and/or eliminate the statistical correlations between rotational and translational parameters that arise as mathematically induced artifacts in other approaches. This paper, together with the statistical analysis in the companion paper for determining the locations of the pivot points, provides everything needed to understand the output of the program as it relates to individual structures.

Base Composition↗
PubMed2000

Multiple functions of an evolutionarily conserved RNA binding domain.

Ribosomal protein L30 of Saccharomyces cerevisiae binds to a distinct RNA structure to inhibit the splicing and the translation of its own transcript. Remarkably, the ortholog of L30 from the archaeon Sulfolobus acidocaldarius binds specifically to the same RNA fragment and inhibits splicing both in vitro and in vivo. Indeed, expression of Sulfolobus L30 in yeast severely reduces growth by limiting production of the endogenous L30. This conservation of binding specificity implies that the target of regulation in the RPL30 transcript mimics a site in the rRNA that has been conserved for more than a billion years. We identify this site, whose location suggests that L30, which has no apparent eubacterial ortholog, is responsible for establishing the orientation of a key bridge between the large and small ribosomal subunits.

Amino Acid Sequence↗
PubMed1997

Subtle atomic group discrimination in the RNA minor groove.

As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.

Amino Acid Sequence↗
PubMed2001

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit.

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.

5' Untranslated Regions↗