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At least 145 records · Page 8Linked to original sources
PubMed2002

A novel mechanism for protein-assisted group I intron splicing.

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site.

Base Sequence↗
PubMed1996

Fluorescence emission of ethidium bromide intercalated in defined DNA duplexes: evaluation of hydrodynamics components.

The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly invoked in single-stranded DNA and RNA tertiary structure. These tertiary structures of nucleic acids are defined by their double stranded regions, and their orientation in the molecular frame constitutes an important component of the nucleic acid structure. A direct view of these tertiary structures can be obtained by fluorescence polarization anisotropy of bound ethidium bromide (EB). The orientation of the dye in the molecular frame of the nucleic acid yields the orientation of the helix. The complete anisotropy function for EB intercalated in genome-derived DNA duplexes was derived by Allison and Schurr (1979) and accounts for base-pair twisting and DNA bending. Single-stranded ribozymes, ribosomal and transfer RNAs, and model DNA junctions contain double-stranded regions shorter than 35 bp in length, for which bending is not significant. We developed and experimentally verified an expression of the anisotropy function for short DNA duplexes which is theoretically compatible with the existing theory, originally developed for long nucleic acids (Schurr et al., 1992). Simulations showed that for DNA duplexes shorter than 35 bp, our expression of the anisotropy function is equivalent to Schurr's and is consistent with experiments carried out on eight DNA duplexes. Modeling the eight duplexes as cylinders, we calculate a duplex diameter of 1.91 +/- 0.15 nm when EB makes a 90 degrees angle with the DNA helix axis and undergoes anisotropic wobbling and 1.97 +/- 0.15 nm when EB makes a 70.5 degrees angle and undergoes isotropic wobbling, respectively. We used this treatment to establish the conformation of five DNA oligonucleotides made of single and tethered hairpins, some designed to exhibit coaxial stacking. Analysis of the fluorescence anisotropy decays shows that the tethered hairpins take an extended rather than parallel conformation. It also shows that the DNA oligonucleotides made of two tethered hairpins exhibit freedom compatible with two independent hairpins. When the linker between hairpins is shortened, the two hairpins are not independent anymore as probed by fluorescence anisotropy, suggesting coaxial stacking of the two helices.

Base Sequence↗
PubMed1991

Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal.

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

Base Sequence↗
PubMed1990

The carboxyl-terminal part of the putative Berne virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionarily related.

Sequence analysis of the 3' part (8 kb) of the polymerase gene of the torovirus prototype Berne virus (BEV) revealed that this area contains at least two open reading frames (provisionally designated ORF1a and ORF1b) which overlap by 12 nucleotides. The complete sequence of ORF1b (6873 nucleotides) was determined. Like the coronaviruses, BEV was shown to express its ORF1b by ribosomal frameshifting during translation of the genomic RNA. The predicted tertiary RNA structure (a pseudoknot) in the toro- and coronaviral frameshift-directing region is similar. Analysis of the amino acid sequence of the predicted BEV ORF1b translation product revealed homology with the ORF1b product of coronaviruses. Four conserved domains were identified: the putative polymerase domain, an area containing conserved cysteine and histidine residues, a putative helicase motif, and a domain which seems to be unique for toro- and coronaviruses. The data on the 3' part of the polymerase gene of BEV supplement previously observed similarities between toro- and coronaviruses at the level of genome organization and expression. The two virus families are more closely related to each other than to other families of positive-stranded RNA viruses.

Amino Acid Sequence↗
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↗
PubMed2005

Long-range RNA-RNA interactions circularize the dengue virus genome.

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

Animals↗
PubMed1992

cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.

Animals↗
PubMed1989

Enzymatic approaches to probing of RNA secondary and tertiary structure.

To make strong statements about possible tertiary structure or the relative stability of regions of secondary structure, the structure-probing experiments must go further than single-hit reactions. Some elements of the environment of the RNA molecule must be altered systematically. Knowledge of the effects of ions or other interacting factors on the activity or physical parameters (e.g., NMR and melting cooperativity) of the RNA help in experimental design. For example, the copious work on tRNA(Phe) compared the crystal and solution structures and allowed the direct correlation of Mg2+ stabilization of the tertiary structure of that molecule. Figure 3 demonstrates that pre-tRNA(Leu-3) responds to Mg2+ depletion in the same manner as detected by the appearance of highly sensitive RNase cleavage sites in the D and T psi C loops. Similar experiments titrating polyamine concentrations suggested that secondary structure was more efficiently stabilized by polyamines than by Mg2+. The variation of Mg2+ concentrations has been used to gain additional information about other RNA structures. Others have used protein-RNA interactions to approach the question of the functional structure of a RNA (for examples, see Ref. 3). Thus, the ideal parameters to choose would be those known to affect the function of the RNA. The variation of Mg2+ and polyamine concentrations would minimally suggest regions of greater or lesser secondary or tertiary structure stability.

Base Sequence↗
PubMed1993

Quaternion representation of RNA sequences and tertiary structures.

A quaternion representation of nucleotides is proposed, with representation of RNA sequences by vectors whose elements are quaternions. Structure and transition matrices in quaternion representation are defined. Correspondence between diagrammatic technique in complex-number and quaternion representation of nucleotides is delineated.

Base Sequence↗
PubMed2001

Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.

5' Untranslated Regions↗
PubMed2004

A guide to ions and RNA structure.

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.

Animals↗
PubMed1977

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.

Binding Sites↗
PubMed2001

Distribution of substitution rates and location of insertion sites in the tertiary structure of ribosomal RNA.

The relative substitution rate of each nucleotide site in bacterial small subunit rRNA, large subunit rRNA and 5S rRNA was calculated from sequence alignments for each molecule. Two-dimensional and three-dimensional variability maps of the rRNAs were obtained by plotting the substitution rates on secondary structure models and on the tertiary structure of the rRNAs available from X-ray diffraction results. This showed that the substitution rates are generally low near the centre of the ribosome, where the nucleotides essential for its function are situated, and that they increase towards the surface. An inventory was made of insertions characteristic of the Archaea, Bacteria and Eucarya domains, and for additional insertions present in specific eukaryotic taxa. All these insertions occur at the ribosome surface. The taxon-specific insertions seem to arise randomly in the eukaryotic evolutionary tree, without any phylogenetic relatedness between the taxa possessing them.

Archaea↗
PubMed1996

Cis-element, oriR, involved in the initiation of (-) strand poliovirus RNA: a quasi-globular multi-domain RNA structure maintained by tertiary ('kissing') interactions.

The key steps in the replication of the poliovirus genome, initiation of (-) and (+) strands, require two different cis-acting elements, oriR and oriL, respectively. It has been proposed that the spatial organization of these elements is maintained by tertiary ('kissing') interactions between the loops of two constituent hairpins. Here, the putative partners of the kissing interaction within the oriR of the full-length poliovirus RNA were modified by site-directed mutagenesis. The destabilization of this interaction resulted in a severe suppression of the viral RNA synthesis, but the mutant transcripts proved to be infectious. With a single exception, the potential for the kissing interaction within the oriR of the recovered viruses was partially or completely restored due to either true reversions or second-site compensatory mutations. There was a good correlation between the restoration of this potential and the phenotypic properties of the viruses. It was concluded that the kissing interaction in the poliovirus oriR is functionally important. Using the above experimental data, a three-dimensional structure was derived by molecular modeling techniques, which demonstrated the overall feasibility of the proposed interactions and displayed the poliovirus oriR as a quasi-globular multi-domain structure.

Animals↗
PubMed1999

Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit.

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967xC1400 completely, increases the frequency of cross-link C1402x1501 twofold, and decreases the cross-link G894xU244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934xU1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

Anti-Bacterial Agents↗
PubMed1991

The novel tertiary structure in delta RNA may function as a ribozyme control element.

The viroid-like domain making up the lefthand end of the delta hepatitis genome (Branch et al., 1989) has structural elements whose interactions may be essential for replication. This portion of the genome is highly conserved in primary sequence and contains two well-defined types of structural features: sites capable of self-cleavage, and those forming a UV-sensitive element of local tertiary structure which is very stable and contains non-Watson:Crick bonds that may form a distinctive surface for binding specific proteins. The proximity of the tertiary structure to the genomic self-cleavage site suggests that the photoreactive element may regulate the ribozyme. This element's stability would limit "breathing" in this region of the circular genome, maintaining the ribozyme in the "off" conformation; its tight structure could be relieved by protein binding at the time of replication. We previously mapped a novel local tertiary structure to a highly conserved portion of the viroid genome (Branch et al., 1985). The many additional properties shared by viroids, related infectious circular RNAs of plants, and the delta agent are discussed in a recent article (Branch et al., in press-a).

Base Sequence↗
PubMed1994

Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

Crystallography↗