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At least 253 records · Page 14Linked to original sources
PubMed2006

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets.

BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and alpha-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two alpha-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

Base Sequence↗
PubMed2004

A probabilistic model for the evolution of RNA structure.

BACKGROUND: For the purposes of finding and aligning noncoding RNA gene- and cis-regulatory elements in multiple-genome datasets, it is useful to be able to derive multi-sequence stochastic grammars (and hence multiple alignment algorithms) systematically, starting from hypotheses about the various kinds of random mutation event and their rates. RESULTS: Here, we consider a highly simplified evolutionary model for RNA, called "The TKF91 Structure Tree" (following Thorne, Kishino and Felsenstein's 1991 model of sequence evolution with indels), which we have implemented for pairwise alignment as proof of principle for such an approach. The model, its strengths and its weaknesses are discussed with reference to four examples of functional ncRNA sequences: a riboswitch (guanine), a zipcode (nanos), a splicing factor (U4) and a ribozyme (RNase P). As shown by our visualisations of posterior probability matrices, the selected examples illustrate three different signatures of natural selection that are highly characteristic of ncRNA: (i) co-ordinated basepair substitutions, (ii) co-ordinated basepair indels and (iii) whole-stem indels. CONCLUSIONS: Although all three types of mutation "event" are built into our model, events of type (i) and (ii) are found to be better modeled than events of type (iii). Nevertheless, we hypothesise from the model's performance on pairwise alignments that it would form an adequate basis for a prototype multiple alignment and genefinding tool.

Evolution, Molecular↗
PubMed2005

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn.

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C'/D' motifs. We show here that conserved terminal stem-loops formed by the C'/D' motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem-loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.

Archaeal Proteins↗
PubMed1984

5S RNA structure and interaction with transcription factor A. 2. Ribonuclease probe of the 7S particle from Xenopus laevis immature oocytes and RNA exchange properties of the 7S particle.

The 5S RNA complexed in the 7S particle of immature Xenopus laevis oocytes was 32P labeled at its 3' end and then subjected in situ to partial digestion using ribonucleases T1, T2, A, and V1 in order to study the conformation of the complexed RNA and its interaction with the transcription factor A (TFIIIA). Digested samples were gel purified to retrieve 5S RNA that was still complexed with the transcription factor protein, and the cleavages in these RNAs were analyzed on sequencing gels. The RNA associated with the 7S particle is very susceptible to ribonuclease activity despite the presence of the protein. Also, the 5S RNA in the 7S particle is in a different conformation from renatured Xenopus laevis (Xlo) 5S RNA and appears to have less secondary structure predominantly in the stem that includes helices IV and V. A species of native Xlo 5S RNA which was isolated from 7S particle preparations under nondenaturing conditions revealed a conformation that was more similar to the 5S RNA in the 7S particle than to renatured 5S RNA. Comparison of data from partial ribonuclease digestions performed on renatured 5S RNA, on the native 5S RNA, and on the complexed 5S RNA allowed us to approximate sites of protein-induced structural change in the complexed 5S RNA, which may signal protein interaction domains. These sites include the 5' side of helices III and V. In another approach to the study of 5S RNA-TFIIIA interactions, we have observed that incubation of 32P-labeled Xlo 5S RNA with 7S particles results in the incorporation of labeled RNA into 7S particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

RNA structure analysis at single nucleotide resolution by selective 2'-hydroxyl acylation and primer extension (SHAPE).

The reactivity of an RNA ribose hydroxyl is shown to be exquisitely sensitive to local nucleotide flexibility because a conformationally constrained adjacent 3'-phosphodiester inhibits formation of the deprotonated, nucleophilic oxyanion form of the 2'-hydroxyl group. Reaction with an appropriate electrophile, N-methylisatoic anhydride, to form a 2'-O-adduct thus can be used to monitor local structure at every nucleotide in an RNA. We develop a quantitative approach involving Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to map the structure of and to distinguish fine differences in structure for tRNAAsp transcripts at single nucleotide resolution. Modest extensions of the SHAPE approach will allow RNA structure to be monitored comprehensively and at single nucleotide resolution for RNAs of arbitrary sequence and structural complexity and under diverse solution environments.

Acylation↗
PubMed1988

Domain VI of Escherichia coli 23 S ribosomal RNA. Structure, assembly and function.

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Autoradiography↗
PubMed2003

RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.

5' Untranslated Regions↗
PubMedDate not supplied

Modulating viral gene expression by aptamers to RNA structures.

Oligonucleotides exhibiting a strong affinity and a high specificity for RNA hairpins were obtained by in vitro selection. Such oligomers give rise to loop-loop complexes with the target hairpins: the trans-activation responsive (TAR) element of the Human Immunodeficiency virus-1 (HIV-1) or subdomains of the Hepatitis C virus (HCV) mRNA. Chemically modified derivatives of an antiTAR aptamer were shown to compete out the binding of the viral protein Tat and to selectively inhibit the in vitro TAR-dependent transcription of a reporter gene. In addition, antisense oligomers derived from sequences selected against the domain IIId of the HCV internal ribosome entry site were shown to specifically block translation both in a cell-free assay and in cultured cells.

Animals↗
PubMed2005

Accelerated probabilistic inference of RNA structure evolution.

BACKGROUND: Pairwise stochastic context-free grammars (Pair SCFGs) are powerful tools for evolutionary analysis of RNA, including simultaneous RNA sequence alignment and secondary structure prediction, but the associated algorithms are intensive in both CPU and memory usage. The same problem is faced by other RNA alignment-and-folding algorithms based on Sankoff's 1985 algorithm. It is therefore desirable to constrain such algorithms, by pre-processing the sequences and using this first pass to limit the range of structures and/or alignments that can be considered. RESULTS: We demonstrate how flexible classes of constraint can be imposed, greatly reducing the computational costs while maintaining a high quality of structural homology prediction. Any score-attributed context-free grammar (e.g. energy-based scoring schemes, or conditionally normalized Pair SCFGs) is amenable to this treatment. It is now possible to combine independent structural and alignment constraints of unprecedented general flexibility in Pair SCFG alignment algorithms. We outline several applications to the bioinformatics of RNA sequence and structure, including Waterman-Eggert N-best alignments and progressive multiple alignment. We evaluate the performance of the algorithm on test examples from the RFAM database. CONCLUSION: A program, Stemloc, that implements these algorithms for efficient RNA sequence alignment and structure prediction is available under the GNU General Public License.

Algorithms↗
PubMed2002

Tracing the evolution of RNA structure in ribosomes.

The elucidation of ribosomal structure has shown that the function of ribosomes is fundamentally confined to dynamic interactions established between the RNA components of the ribosomal ensemble. These findings now enable a detailed analysis of the evolution of ribosomal RNA (rRNA) structure. The origin and diversification of rRNA was studied here using phylogenetic tools directly at the structural level. A rooted universal tree was reconstructed from the combined secondary structures of large (LSU) and small (SSU) subunit rRNA using cladistic methods and considerations in statistical mechanics. The evolution of the complete repertoire of structural ribosomal characters was formally traced lineage-by-lineage in the tree, showing a tendency towards molecular simplification and a homogeneous reduction of ribosomal structural change with time. Character tracing revealed patterns of evolution in inter-subunit bridge contacts and tRNA-binding sites that were consistent with the proposed coupling of tRNA translocation and subunit movement. These patterns support the concerted evolution of tRNA-binding sites in the two subunits and the ancestral nature and common origin of certain structural ribosomal features, such as the peptidyl (P) site, the functional relay of the penultimate stem helix of SSU rRNA, and other structures participating in ribosomal dynamics. Overall results provide a rare insight into the evolution of ribosomal structure.

Animals↗
PubMedDate not supplied

From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements.

Structural elements in the 5' region of a bacterial mRNA can have major effects on expression of downstream coding sequences. Folding of the nascent RNA into the helix of an intrinsic transcriptional terminator results in premature termination of transcription and in failure to synthesize the full-length transcript. Structure in the translation initiation region of an mRNA blocks access of the translation initiation complex to the ribosome binding site, thereby preventing protein synthesis. RNA structures can also affect the stability of an RNA by altering sensitivity to ribonucleases. A wide variety of mechanisms have been uncovered in which changes in mRNA structure in response to a regulatory signal are used to modulate gene expression in bacteria. These systems allow the cell to recognize an impressive array of signals, and to monitor those signals in many different ways.

DNA-Directed RNA Polymerases↗
PubMed2006

Efficient pairwise RNA structure prediction and alignment using sequence alignment constraints.

BACKGROUND: We are interested in the problem of predicting secondary structure for small sets of homologous RNAs, by incorporating limited comparative sequence information into an RNA folding model. The Sankoff algorithm for simultaneous RNA folding and alignment is a basis for approaches to this problem. There are two open problems in applying a Sankoff algorithm: development of a good unified scoring system for alignment and folding and development of practical heuristics for dealing with the computational complexity of the algorithm. RESULTS: We use probabilistic models (pair stochastic context-free grammars, pairSCFGs) as a unifying framework for scoring pairwise alignment and folding. A constrained version of the pairSCFG structural alignment algorithm was developed which assumes knowledge of a few confidently aligned positions (pins). These pins are selected based on the posterior probabilities of a probabilistic pairwise sequence alignment. CONCLUSION: Pairwise RNA structural alignment improves on structure prediction accuracy relative to single sequence folding. Constraining on alignment is a straightforward method of reducing the runtime and memory requirements of the algorithm. Five practical implementations of the pairwise Sankoff algorithm - this work (Consan), David Mathews' Dynalign, Ian Holmes' Stemloc, Ivo Hofacker's PMcomp, and Jan Gorodkin's FOLDALIGN - have comparable overall performance with different strengths and weaknesses.

Algorithms↗
PubMed1999

Force field based conformational analysis of RNA structural motifs: GNRA tetraloops and their pyrimidine relatives.

The protocol of conformational analysis applied here to ribonucleotide oligomers combines conformational search in the space of torsion angles and energy minimization using the AMBER4.1 force field with a continuum treatment of electrostatic solute-solvent interactions. RNA fragments with 5'-GGGCGNNAGCCU-3' sequences commonly fold into hairpins with four-membered loops. The combinatorial search for acceptable conformations using the MC-SYM program was restricted to loop nucleotides and yielded roughly 1500 structures being compatible with a double-stranded stem. After energy minimization by the JUMNA program (without applying any experimental constraints), these structures converged into an ensemble of 74 different conformers including 26 structures which contained the sheared G-A base pair observed in experimental studies of GNRA tetraloops. Energetic analysis shows that inclusion of solvent electrostatic effects is critically important for the selection of conformers that agree with experimentally determined structures. The continuum model accounts for solvent polarization by means of the electrostatic reaction field. In the case of GNRA loop sequences, the contributions of the reaction field shift relative stabilities towards conformations showing most of the structural features derived from NMR studies. The agreement of computed conformations with the experimental structures of GAAA, GCAA, and GAGA tetraloops suggests that the continuum treatment of the solvent represents a definitive improvement over methods using simple damping models in electrostatic energy calculations. Application of the procedure described here to the evaluation of the relative stabilities of conformers resulting from searching the conformational space of RNA structural motifs provides some progress in (non-homology based) RNA 3D-structure prediction.

Base Sequence↗
PubMed1993

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Affinity Labels↗
PubMed1985

Comparison of eubacterial and eukaryotic 5S RNA structures: a chemical modification study.

The 5S RNAs from Bacillus stearothermophilus and Saccharomyces cerevisiae were probed by nucleotide-specific reagents, with a view to compare and contrast their higher order structures. The progressive unfolding of the RNAs during heating, in the presence and absence of magnesium, was monitored. Evidence was provided for the double-helical segments which occur in the secondary structural models of both RNAs. The results also placed constraints on the possible structuring of the remainder of the RNA and yielded some insight into ways of folding up the molecule. Together with the data from our earlier studies, employing ribonucleases, these results provide a detailed picture of the structuring and topography of the 5S RNAs. The main structural differences between the eubacterial and eukaryotic RNAs occur throughout the loop D/helix IV/loop E/helix V arm; in particular strong evidence is provided for loop D of the eukaryotic RNA being involved in a tertiary interaction.

Geobacillus stearothermophilus↗
PubMed1988

Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing.

The structure and structural transitions of single-stranded RNA were investigated by energy calculations and temperature-gradient gel electrophoresis. Most experiments have been carried out on RNA of mature viroids and their replication intermediates, which are RNA (-) strand oligomers and RNA (+) strand oligomers. The technique of temperature-gradient gel electrophoresis proved to be particularly useful for analysing co-existing structures. The secondary structure of lowest free energy for unit length and oligomeric replication intermediates is an extended rod-like structure similar to that of the mature circular viroid. When this structure is used as a model for calculations, there is a large degree of agreement between theoretical and experimental curves. Under particular solution conditions, however, (+) strand oligomers undergo a rearrangement from the extended structure to a branched structure, in which every two units form a region of three helices, together 28 bp long. This structure is called the tri-helical structure. The process of structure formation during the synthesis of oligomers could be followed: at first, a transient multi-branched structure is formed which is then transformed into the extended and the tri-helical structures. The region of the three stable helices serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes, and be processed into circular mature viroids. Co-transcription of complementary (+) and (-) strands shows that energetically favored double-strand formation may at least partially be prohibited by stable secondary structures of the single strands. Natural replication intermediates have been analysed in respect to their subcellular location and their size distribution. They are associated with the nucleoli as was found earlier for mature viroids. Natural (-) strand oligomers are larger than (+) strand oligomers; both types show a periodicity in the size distribution of two units. The models of the structures, which are involved in viroid processing, are in accordance with recent infectivity data and with the results on natural replication intermediates.

Base Sequence↗
PubMed1986

RNA structural elements for expression in Escherichia coli. Alpha 1-antitrypsin synthesis using translation control elements based on the cII ribosome-binding site of phage lambda.

Analysis of a series of lambda cII::alpha 1-antitrypsin (alpha 1AT) gene fusions of different sizes showed that increased alpha 1AT expression correlated with the stabilisation of a particular computer-predicted RNA secondary structure. Moreover, significant synthesis of unfused alpha 1AT was achieved by reconstruction of this conformation to permit interaction between the upstream region of the ribosome-binding site and the first part of the alpha 1AT coding sequence. This high-level expression was dependent upon certain silent point mutations in the coding sequence, indicating that RNA primary and secondary structure determinants can operate in concert to dictate the efficiency of protein synthesis.

Bacteriophage lambda↗