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At least 1,315 records · Page 73Linked to original sources
PubMed2005

A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

Bromovirus↗
PubMed1988

The 9S RNA precursor of Escherichia coli 5S RNA has three structural domains: implications for processing.

The secondary structure of the 9S RNA precursor to ribosomal 5S RNA in Escherichia coli has been determined using chemical reagents and ribonucleases in combination with a reverse transcription procedure. The 9S RNA precursor was generated in vitro by T7 RNA polymerase, and the rrnB operon terminator, T1, was able to terminate the in vitro transcript. The secondary structure model exhibits three structural domains corresponding to a 5' region, a mature region and a terminator region. The mature domain is structurally identical to 5S RNA, and the ribosomal proteins L18 and L25 are able to bind to the precursor. The processing endoribonuclease RNase E cleaves between the structural domains. Moreover, an intramolecular refolding of the nascent transcript must take place if the current view of RNase III processing stems is correct.

Cloning, Molecular↗
PubMed1999

Comparison of the crystal and solution structures of two RNA oligonucleotides.

Until recently, there were no examples of RNAs whose structures had been determined by both NMR and x-ray crystallography, and thus there was no experimental basis for assessing the accuracy of RNA solution structures. A comparison of the solution and the crystal structures of two RNAs is presented, which demonstrates that NMR can produce solution structures that resemble crystal structures and thus validates the application to RNA of a methodology developed initially for the determination of protein conformations. Models for RNA solution structures are appreciably affected by the parameters used for their refinement that describe intramolecular interactions. For the RNAs of interest here, the more realistic those parameters, the greater the similarity between solution structures and crystal structures.

Base Sequence↗
PubMed1994

Crystal and molecular structure of r(CGCGAAUUAGCG): an RNA duplex containing two G(anti).A(anti) base pairs.

BACKGROUND: Non-Watson-Crick base pair associations contribute significantly to the stabilization of RNA tertiary structure. The conformation adopted by such pairs appears to be a function of both the sequence and the secondary structure of the RNA molecule. G.A mispairs adopt G(anti).A(anti) configurations in some circumstances, such as the ends of helical regions of rRNAs, but in other circumstances probably adopt an unusual configuration in which the inter-base hydrogen bonds involve functional groups from other bases. We investigated the structure of G.A pairs in a synthetic RNA dodecamer, r(CGCGAAUUAGCG), which forms a duplex containing two such mismatches. RESULTS: The structure of the RNA duplex was determined by single crystal X-ray diffraction techniques to a resolution in the range 7.0-1.8A, and found to be an A-type helical structure with 10 Watson-Crick pairs and two G.A mispairs. The mispairs adopt the G(anti).A(anti) conformation, held together by two obvious hydrogen bonds. Unlike analogous base pairs seen in a DNA duplex, they do not exhibit a high propeller twist and may therefore be further stabilized by weak, reverse, three-center hydrogen bonds. CONCLUSIONS: G(anti).A(anti) mispairs are held together by two hydrogen of guanine and the N6 and N1 of adenine. If the mispairs do not exhibit high propeller twist they may be further stabilized by inter-base reverse three-centre hydrogen bonds. These interactions, and other hydrogen bonds seen in our study, may be important in modelling the structure of RNA molecules and their interactions with other molecules.

Animals↗
PubMed2006

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits.

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.

Deinococcus↗
PubMed1989

Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P.

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.

Bacillus subtilis↗
PubMed2000

Structural genomics of RNA.

A detailed understanding of the functions and interactions of biological macromolecules requires knowledge of their molecular structures. Structural genomics, the systematic determination of all macromolecular structures represented in a genome, is focused at present exclusively on proteins. It is clear, however, that RNA molecules play a variety of significant roles in cells, including protein synthesis and targeting, many forms of RNA processing and splicing, RNA editing and modification, and chromosome end maintenance. To comprehensively understand the biology of a cell, it will ultimately be necessary to know the identity of all encoded RNAs, the molecules with which they interact and the molecular structures of these complexes. This report focuses on the feasibility of structural genomics of RNA, approaches to determining RNA structures and the potential usefulness of an RNA structural database for both predicting folds and deciphering biological functions of RNA molecules.

Computational Biology↗
PubMed1981

Secondary structure of 16S ribosomal RNA.

A secondary structure model for 16S ribosomal RNA which is based on available chemical, enzymatic, and comparative sequence data shows good agreement between constraints dictated by the model and a wide variety of experimental observations. The four major structural domains created by the base-pairing scheme correspond closely to RNA fragments isolated after nuclease digestion in the presence of bound ribosomal proteins. Functionally important sites appear to be located in unpaired regions and are phylogenetically highly conserved.

Base Sequence↗
PubMed2001

Mechanism of ribosome recruitment by hepatitis C IRES RNA.

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Animals↗
PubMed2002

Novel paromamine derivatives exploring shallow-groove recognition of ribosomal-decoding-site RNA.

Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.

Aminoglycosides↗
PubMed2003

NMR spectroscopy of RNA.

NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base-pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.

Base Pairing↗
PubMed2000

Specific interaction between the hepatitis delta virus RNA and glyceraldehyde 3-phosphate dehydrogenase: an enhancement on ribozyme catalysis.

Replication of hepatitis delta virus (HDV) RNA occurs in the nuclei of infected cells. The replication is mediated by cellular factors containing an RNA polymerase II-like enzyme activity through a double rolling-circle mechanism and is regulated by delta antigens. In this study, UV cross-linking experiments were carried out to examine interactions between HDV RNA and proteins present in HeLa nuclear extract. Cellular proteins with molecular mass of 23 (p23), 36 (p36), 38 (p38), and 58 (p58) kDa bound to full-length HDV RNA of both genomic and antigenomic strands. Deletion analysis on the antigenomic strand mapped the interacting domain within a 79-nucleotide fragment but not at the ends of the rod-shaped viral RNA structure. The specificity of the RNA-protein interactions was demonstrated by competition experiments and the specific HDV RNA-binding proteins were purified through column chromatography. Electrophoresis mobility shift assay with the purified fractions demonstrated that the interaction between p36 and HDV RNA was relatively stable even in the presence of 0.5 M NaCl. Biochemical analysis including protein microsequencing identified the p36 as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RNase footprinting indicated that the UC-rich domain between nucleotides 379 and 414 of the HDV antigenomic RNA was involved in the GAPDH binding. Functional studies further demonstrated an enhancing effect of GAPDH on the ribozyme activity of HDV antigenomic RNA. In addition, in the presence of HDV RNA cellular GAPDH relocalized from the cytoplasm to the nucleus where HDV replication occurs. These results suggest that GAPDH is involved in the replication of HDV.

Amino Acid Sequence↗
PubMed1998

Amino acids as RNA ligands: a direct-RNA-template theory for the code's origin.

Numerous RNA binding sites for specific amino acids are now known, coming predominantly from selection-amplification experiments. These sites are chemically discriminating despite being predominantly small, simple RNA structures: internal and bulge loops. Recent studies of sites for hydrophobic side chains suggest that there are other generalizable structural features which recur in hydrophobic RNA sites. Further, sites for hydrophobic side chains can contain codons for the bound amino acid, as has also long been known for the polar amino acid arginine. Such findings are comprehensively reviewed, and the implications for the origin of coded peptide synthesis are considered. An origins hypothesis which accommodates all the data, DRT (direct RNA templating), is formulated.

Amino Acids↗
PubMed1988

Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation.

The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases. We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro. Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E. The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs. Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A. These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.

Autoradiography↗
PubMed2001

Synthesis and analysis of RNA containing 6-trifluoromethylpurine ribonucleoside.

We report the synthesis of a 5'-DMT-2'-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside ((TFM)P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of (TFM)P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases. Reaction: see text.

Amides↗
PubMed1999

Helicase motifs: the engine that powers DNA unwinding.

Helicases play essential roles in nearly all DNA metabolic transactions and have been implicated in a variety of human genetic disorders. A hallmark of these enzymes is the existence of a set of highly conserved amino acid sequences termed the 'helicase motifs' that were hypothesized to be critical for helicase function. These motifs are shared by another group of enzymes involved in chromatin remodelling. Numerous structure-function studies, targeting highly conserved residues within the helicase motifs, have been instrumental in uncovering the functional significance of these regions. Recently, the results of these mutational studies were augmented by the solution of the three-dimensional crystal structure of three different helicases. The structural model for each helicase revealed that the conserved motifs are clustered together, forming a nucleotide-binding pocket and a portion of the nucleic acid binding site. This result is gratifying, as it is consistent with structure-function studies suggesting that all the conserved motifs are involved in the nucleotide hydrolysis reaction. Here, we review helicase structure-function studies in the light of the recent crystal structure reports. The current data support a model for helicase action in which the conserved motifs define an engine that powers the unwinding of duplex nucleic acids, using energy derived from nucleotide hydrolysis and conformational changes that allow the transduction of energy between the nucleotide and nucleic acid binding sites. In addition, this ATP-hydrolysing engine is apparently also associated with proteins involved in chromatin remodelling and provides the energy required to alter protein-DNA structure, rather than duplex DNA or RNA structure.

Adenosine Triphosphate↗
PubMed1992

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.

Base Sequence↗