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At least 1,729 records · Page 96Linked to original sources
PubMed1988

Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA.

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.

Chemical Phenomena↗
PubMed2004

Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.

Initiation of translation in eukaryotic cells can occur by two distinct mechanisms, cap-dependent scanning and internal ribosome entry. The latter mechanism requires the formation of a complex RNA structural element termed an internal ribosome entry segment (IRES). IRESs are located in the 5' untranslated region of the message, and in the presence of trans-acting factors allow the ribosome to be recruited to a site that is a considerable distance from the cap structure. Many cellular mRNAs have now been shown to contain IRESs and it is likely that up to 10% of all mRNAs have the capability to initiate translation by this mechanism. The majority of IRESs that have been identified thus far are found in mRNAs whose protein products are associated with the control of cell growth and cell death, including many growth factors, proto-oncogenes and proteins required for apoptosis. In this review, we discuss the cellular situations when IRESs are required, the trans-acting factors that are necessary for IRES function and deregulation of IRES-mediated translation in tumorigenesis.

Animals↗
PubMed2001

Functional equivalence of the uridine turn and the hairpin as building blocks of tertiary structure in the Neurospora VS ribozyme.

Mutational, kinetic, and chemical modification experiments show that one of the three-way helical junctions in the Neurospora VS ribozyme contains a uridine turn that is important for organizing the functional three-dimensional structure of this junction. Disruption of the uridine turn disrupts the structure of the junction and decreases the self-cleavage activity of the ribozyme; however, substitution of the uridine turn with a variety of different hairpins, thereby transforming the three-way junction into a four-way junction, maintains catalytic activity. Chemical modification structure probing reveals that both the native junction and the hairpin-containing junction support the same tertiary interactions required elsewhere in the ribozyme for catalysis. These observations show that functionally equivalent three-dimensional RNA structures can be built from different secondary structure elements.

Base Sequence↗
PubMed1994

The Escherichia coli ribosomal RNA leader: a structural and functional investigation.

The structure of the E. coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification. The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model. The binding of purified Nus proteins to the ribosomal leader RNA was further tested. Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC. The possible significance of this interaction is discussed. In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo. A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant). It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared. High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions. Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation. The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.

Bacterial Proteins↗
PubMed1996

Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA.

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.

Amino Acid Sequence↗
PubMed1995

Translational suppression by trinucleotide repeat expansion at FMR1.

Fragile X syndrome is the result of the unstable expansion of a trinucleotide repeat in the 5'-untranslated region of the FMR1 gene. Fibroblast subclones from a mildly affected patient, each containing stable FMR1 alleles with 57 to 285 CGG repeats, were shown to exhibit normal steady-state levels of FMR1 messenger RNA. However, FMR protein was markedly diminished from transcript with more than 200 repeats. Such transcripts were associated with stalled 40S ribosomal subunits. These results suggest that a structural RNA transition beyond 200 repeats impedes the linear 40S migration along the 5'-untranslated region. This results in translational inhibition by trinucleotide repeat expansion.

Centrifugation, Density Gradient↗
PubMed1994

The thumb's knuckle. Flexibility in the thumb subdomain of T7 RNA polymerase is revealed by the structure of a chimeric T7/T3 RNA polymerase.

We have solved the structure of a chimeric T7/T3 RNA polymerase (RNAP) in an orthorhombic crystal by molecular replacement with the T7 RNAP structure determined from a monoclinic crystal. The structure of the protruding "thumb" subdomain of the polymerase appears very different in these two crystals apparently because of differences in packing contacts made by the thumb subdomain. These observations support the proposal that the thumb subdomain is flexible and can wrap around bound template to obstruct polymerase: template dissociation during processive synthesis.

Amino Acid Sequence↗
PubMed2000

Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation.

For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods. We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme. The cooperativity of folding, m-value, and the free energy, DeltaG degrees N-U, associated with formation of each tertiary contact was determined by analysis of the isotherms. The DeltaG degrees N-U values measured in this study vary from 1.7 +/- 0.2 to 7. 6 +/- 1.2 kcal mol-1. Thus, the stability of these discrete tertiary contacts vary by almost 104. In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol-1 M-1) indicating that these contacts exhibit global cooperatively in their folding behavior. This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.

Allosteric Site↗
PubMed2005

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.

Computer Simulation↗
PubMed1998

Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution.

The crystal structure, at 2.8 A resolution, of an RNA aptamer bound to bacteriophage MS2 coat protein has been determined. It provides an opportunity to compare the interactions of MS2 coat protein and wild type operator with those of an aptamer, whose secondary structure differs from the wild type RNA in having a three-base loop (compared to a tetraloop) and an additional base pair between this loop and the sequence-specific recognition element in the stem. The RNA binds in the same location on the coat protein as the wild type operator and maintains many of the same RNA-protein interactions. In order to achieve this, the RNA stem loop undergoes a concerted rearrangement of the 3' side while leaving the 5' side and the loop interactions largely unchanged, illustrating the ability of RNA to present similar molecular recognition surfaces from distinct primary and secondary structures.

Asparagine↗
PubMed1997

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗
PubMed2003

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

5' Untranslated Regions↗
PubMed2006

The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research.

BACKGROUND: Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs. DESCRIPTION: The Subviral RNA database is a web-based environment that facilitates the research and analysis of viroids, satellite RNAs, satellites viruses, the human hepatitis delta virus, and related RNA sequences. It integrates a large number of Subviral RNA sequences, their respective RNA motifs, analysis tools, related publication links and additional pertinent information (ex. links, conferences, announcements), allowing users to efficiently retrieve and analyze relevant information about these small RNA agents. CONCLUSION: With its design, the Subviral RNA Database could be considered as a fundamental building block for the study of these related RNAs. It is freely available via a web browser at the URL: http://subviral.med.uottawa.ca.

Base Sequence↗
PubMed1996

Exon circularization in mammalian nuclear extracts.

Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

Base Sequence↗
PubMed1996

Two secondary structures for the RRE of HIV-1?

The RRE, the target sequence for the Rev protein of HIV-1, is a highly structured RNA sequence characterized by multiple stem loops. Although agreement on the stem/loop organization outside the high-affinity site was reached some time ago by several laboratories, recent work has suggested an alternative structure in which sequences from two of the stem/loops (IV and V) pair to form one long stem/loop (IV/V) when enough HIV material is present to allow the formation of an extended central stem structure (I/I'). Here we address the contribution of the original and alternative structures to function in RRE constructs with short and extended I'I' regions. We confirm that extended I/I' structures improve RRE function and may stabilize the overall structure. However, we find no evidence that an extended I/I' structure preferentially stabilizes either alternative structure with respect to the other. The two alternative structures are approximately functionally equivalent, and both are probably present in an in vivo population of RREs.

Animals↗
PubMed2004

A conserved element in the yeast RNase MRP RNA subunit can participate in a long-range base-pairing interaction.

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses a putatively catalytic RNA subunit, structurally related to that of RNase P. A thorough structure analysis of Saccharomyces cerevisiae MRP RNA, entailing enzymatic and chemical probing, mutagenesis and thermal melting, identifies a previously unrecognised stem that occupies a position equivalent to the P7 stem of RNase P. Inclusion of this P7-like stem confers on yeast MRP RNA a greater degree of similarity to the core RNase P RNA structure than that described previously and better delimits domain 2, the proposed specificity domain. The additional stem is created by participation of a conserved sequence element (ymCR-II) in a long-range base-pairing interaction. There is potential for this base-pairing throughout the known yeast MRP RNA sequences. Formation of a P7-like stem is not required, however, for the pre-rRNA processing or essential function of RNase MRP. Mutants that can base-pair are nonetheless detrimental to RNase MRP function, indicating that the stem will form in vivo but that only the wild-type pairing is accommodated. Although the alternative MRP RNA structure described is clearly not part of the active RNase MRP enzyme, it would be the more stable structure in the absence of protein subunits and the probability that it represents a valid intermediate species in the process of yeast RNase MRP assembly is discussed.

Base Pairing↗
PubMedDate not supplied

Primary structure of the receptor for calcium channel blockers from skeletal muscle.

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.

Amino Acid Sequence↗
PubMed1991

RNA viruses: genome structure and evolution.

The explosive pace of sequencing of RNA viruses is leading to rapid advances in our understanding of the evolution of these viruses and of the ways in which their genomes are organized and expressed. New insights are coming not only from genomic nucleotide sequence comparisons, but also from direct sequencing of transcribed mRNAs and of RNAs that serve as intermediates in replication.

Biological Evolution↗