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At least 1,621 records · Page 90Linked to original sources
PubMed2001

Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures.

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 microm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.

Blotting, Western↗
PubMedDate not supplied

[Study of the secondary and tertiary structure of phage MS2 RNA using nucleases and fluorescent dyes specific for secondary structure].

The binding of ethidium bromide (EtBr) and acridine orange (AO) to RNA in native state or after hydrolysis by S1 and SV nucleases that specifically split single-stranded and double-stranded segments was studied. Nuclease S1 hydrolysis of RNA does not increase the number of EtBr strong binding sites, Tm and hyperchromic effect being also unchanged. Hydrolysis by double-stranded segments accessible to EtBr is followed by the diminishing of Tm and hyperchromism. A supposition is put forward that the main role in stabilization of the RNA tertiary structure is played by double-stranded segments arranged so that some of them are hidden and do not interact with dyes. One of the possible models may be parallel oriented intramolecular "hair-pins" forming compact "rod-like" structures.

Coliphages↗
PubMed2001

Protein-RNA interactions: a structural analysis.

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.

Base Pairing↗
PubMed2003

2'-mercaptonucleotide interference reveals regions of close packing within folded RNA molecules.

The 2'-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2'-hydroxyl group, we synthesized 2'-mercaptonucleotides for use in nucleotide analogue interference mapping. To correlate the observed interference effects with tertiary structure, we used the independently folding DeltaC209 P4-P6 domain from the Tetrahymena group I intron. We generated populations of DeltaC209 P4-P6 molecules containing 2'-mercaptonucleotides located randomly throughout the domain and separated the folded molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of the RNA molecules revealed the sites at which 2'-mercaptonucleotides interfere with folding. These interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2'-mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed regions within folded RNA molecules.

Models, Molecular↗
PubMed1995

Complete nucleotide sequences of 5'- and 3'-noncoding regions of both genome segments of different strains of infectious bursal disease virus.

The terminal sequences of both genomic dsRNA segments of different strains of infectious bursal disease virus were determined. Analysis of the linear sequences revealed in the 5'-noncoding region of both segments the presence of a 32-nucleotide sequence with motifs conserved between segments and serotypes. The 3'-noncoding regions showed a high degree of conservation within segment A or B, but differed between segments. Different inverted terminal repeats were detected in both segments. A sequence of 13 nucleotides found in the 5'-noncoding regions of both segments could function as a binding site for chicken 18 S rRNA. Secondary structure prediction of the different noncoding regions showed a high degree of similarity. Surprisingly, the predicted secondary structure of the 5'-noncoding region of segment A differed between serotypes. A possible influence of these different RNA structures on viral replication is discussed.

Animals↗
PubMed2004

Molecular basis of box C/D RNA-protein interactions; cocrystal structure of archaeal L7Ae and a box C/D RNA.

We have determined and refined a crystal structure of the initial assembly complex of archaeal box C/D sRNPs comprising the Archaeoglobus fulgidus (AF) L7Ae protein and a box C/D RNA. The box C/D RNA forms a classical kink-turn (K-turn) structure and the resulting protein-RNA complex serves as a distinct platform for recruitment of the fibrillarin-Nop5p complex. The cocrystal structure confirms previously proposed secondary structure of the box C/D RNA that includes a protruded U, a UU mismatch, and two sheared tandem GA base pairs. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct K-turn RNAs revealed a set of remarkably conserved principles in protein-RNA interactions. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.

Amino Acid Sequence↗
PubMed2000

Enzymatic properties of hepatitis C virus NS3-associated helicase.

The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3' to 5' direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem-loop RNA structure (SL I) within the 3'-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.

3' Untranslated Regions↗
PubMed2002

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

Base Sequence↗
PubMed1981

The structural basis of biological systems -- on the structures of transfer RNA and DNA and on some biological consequences.

Tetrahelix polynucleotide structure is important in biological systems. Though it is disputed there is firm evidence that transfer RNA has a tetrahelix structure and has the form of a simple cylinder. There is overwhelming chemical, biochemical and sequence evidence and the structure satisfies the constraints of the unit cell lattice. Tetrahelix structure also occurs in DNA and appears to be the most stable form and in many cases the naturally occurring form under physiological conditions. The consequences of tetrahelix structure clarify some longstanding problems such as the physical basis of supertwisting and of compact structure.

Codon↗
PubMed2000

Transcription and replication of the influenza a virus genome.

The genome of influenza A virus consists of eight segments of negative-strand viral RNA (vRNA). During the replication cycle of the virus, the genomic vRNA is transcribed into positive-strand mRNA and complementary RNA (cRNA) in the cell nucleus. The promoter for the synthesis of mRNA molecules is located in a partially double-stranded RNA structure formed by the 5'- and 3'-terminal sequences of genomic vRNA segments. The virus encoded RNA-dependent RNA polymerase complex has to interact with both ends of the vRNA in order to generate capped RNA primers by endonucleolytic cleavage of cellular pre-mRNAs for the initiation of viral mRNA synthesis. Conserved sequence elements in the 5'-end, e.g. a polymerase binding site and a U(5-7) sequence are required for polyadenylation of virus-specific mRNAs. Polyadenylation occurs by reiterative copying of the U(5-7) sequence by the viral RNA polymerase, which is bound to the 5'end of the vRNA template. The U(5-7) sequence acts directly as a template for the poly(A)-tail. During the replication cycle of the virus, a "switch" from mRNA to cRNA synthesis occurs, but the mechanism by which this switch occurs remains unclear. The viral nucleoprotein and its interaction with the polymerase proteins and vRNA might play a role in this process. In contrast to transcription, the process of replication--the synthesis of cRNA and vRNA, which are known to occur in the absence of primers--is poorly understood.

Animals↗
PubMed2006

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets.

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.

Amino Acid Sequence↗
PubMed1987

Effect of beta-decay of radionuclides incorporated into influenza virus RNA and proteins on the infectivity of the virus and antigenicity of its nucleoprotein.

The effect of beta-decay of radionuclides incorporated into influenza virus on the properties of the two closely adjacent structures--RNA and nucleoprotein (NP)--was studied. The long-term storage of 3H-uridine labelled influenza virus was shown to lead to the loss of infectivity. This effect may be explained by lethal intra-molecular modifications of viral RNA, caused by beta-decay of 3H incorporated into the molecule. There was an accompanying decrease of monoclonal antibody (MAB) binding activity, this also being a plausible result of beta-decay. The different rates of inactivation of MAB binding activity of different epitopes of NP of the 3H-labelled virus shown in our studies suggest that there are different types of structural organization or different location of these epitopes in the NP. The effect of 3H-decay on the intracellular RNA of reproducing virus lead to a decrease in virus yield; this may be due to radiation- and transmutation-induced damage of messenger and progeny RNA populations synthesized during the infection. The storage of influenza virus labelled with 14C-aminoacids lead to a decrease in MAB binding activity of the NP that was unaccompanied by a decrease in infectivity. Furthermore, 14C-decay in proteins of reproducing virus had no adverse effect.

Animals↗
PubMed1993

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.

Animals↗
PubMed1999

Crystal structure of an RNA 16-mer duplex R(GCAGAGUUAAAUCUGC)2 with nonadjacent G(syn).A+(anti) mispairs.

G.A mispairs are one of the most common noncanonical structural motifs of RNA. The 1.9 A resolution crystal structure of the RNA 16-mer r(GCAGAGUUAAAUCUGC)2 has been determined with two isolated or nonadjacent G.A mispairs. The molecule crystallizes with one duplex in the asymmetric unit in space group R3 and unit cell dimensions a = b = c = 49.24 A and alpha = beta = gamma = 51.2 degrees. It is the longest known oligonucleotide duplex at this resolution and isomorphous to the 16-mer duplex with the C.A+ mispairs [Pan, et al., (1998) J. Mol. Biol. 283, 977-984]. The C.A+ mispair behaves like a wobble pair while the G.A+ does not. The G.A mispairs are protonated at N1 of the adenines as in the C.A+ mispairs, and two hydrogen bonds in the G(syn).A+(anti) conformation are formed. The syn guanine is stabilized by an intranucleotide hydrogen bond between the 2-amino and the 5'-phosphate groups. The G(syn).A+(anti) conformation can provide a different surface for recognition in the grooves compared to other G.A hydrogen bonding schemes. The major groove is widened between the two mispairs allowing access to ligands. One of the 3-fold axes is occupied by a sodium ion and a water molecule, while a second is occupied by another water molecule.

Adenine Nucleotides↗
PubMed1999

Prediction and visualization of structural switches in RNA.

There are various cases where the biological function of an RNA molecule involves a reversible change of conformation. paRNAss is a software approach to the prediction of such structural switching in RNA. It is based on three hypotheses about the secondary structure space of a switching RNA molecule, which can be evaluated by RNA folding and structure comparison. In the positive case, the predicted structures must be verified experimentally. Additionally, we give an animated visualization of an energetically favourable transition between the predicted structures. paRNAss is available via the Bielefeld Bioinformatics Server. This paper explains the underlying model and shows that the approach performs well in a variety of applications.

Base Sequence↗
PubMed2004

The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.

Introns↗
PubMed1998

Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta.

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.

Allolevivirus↗
PubMed2003

Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme.

Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are "brittle," breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

Animals↗