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At least 631 records · Page 35Linked to original sources
PubMed1995

Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage.

The alpha subunit of Escherichia coli RNA polymerase plays essential roles in protein-protein contacts, not only for RNA polymerase assembly, but also for transcription activation by class I factors. To reveal the structure-function relationship of the alpha subunit, we attempted to elucidate the organization of the structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. The results indicate that one region, Arg235 to Glu244, is highly accessible to endoproteases. We propose that the alpha subunit consists of two major structural domains, the amino-terminal domain upstream from Arg235 and the carboxy-terminal domain downstream from Glu245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement with its functional map, i.e., the amino-terminal subunit assembly determinants and the carboxy-terminal transcription activation determinants, including the contact sites with class I transcription factors and DNA UP (enhancer) elements. The secondary proteolytic cleavage sites were also determined, in order to analyse intra-domain structures.

Amino Acid Sequence↗
PubMed2002

Biochemical identification of A-minor motifs within RNA tertiary structure by interference analysis.

A-minor motifs are the most common tertiary structural elements in RNA helix packing. Biochemical identification of these interactions is now feasible using interference mapping analysis with the adenosine analogues 2'-deoxyadenosine and 3-deaza-adenosine. This approach was used to demonstrate that A-minor motifs mediate helix packing interactions that are important for 5'-splice site selection in the group I intron. By analysing the interference pattern of several analogues it is possible to identify and distinguish the four variants of the A-minor motif.

Binding Sites↗
PubMed2000

NMR study of a novel RNA quadruplex structure.

The structure of an RNA oligomer, r (GGAGGUUUUGGAGG) (R14-2) whose G-G steps are separated by adenine and uracil residues has been investigated by NMR. In the presence of 20 mM K+, a novel dimeric multiplex architecture is adopted by two strands of R14-2. In each strand a UUUU loop and two A residues connect four parallel G-G steps that pair-align into two tetrads. One of the tetrads is further pair-aligned by two A residues through the sheared mismatch and a novel hexad is subsequently formed. Two hexads coming from two different strands stack to make a dimeric multiplex. All of the guanosine and adenosine residues take an anti conformation.

Base Sequence↗
PubMed2006

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Animals↗
PubMed1997

ESSA: an integrated and interactive computer tool for analysing RNA secondary structure.

With ESSA, we propose an approach of RNA secondary structure analysis based on extensive viewing within a friendly graphical interface. This computer program is organized around the display of folding models produced by two complementary methods suitable to draw long RNA molecules. Any feature of interest can be managed directly on the display and highlighted by a rich combination of colours and symbols with emphasis given to structural probe accessibilities. ESSA also includes a word searching procedure allowing easy visual identification of structural features even complex and degenerated. Analysis functions make it possible to calculate the thermodynamic stability of any part of a folding using several models and compare homologous aligned RNA both in primary and secondary structure. The predictive capacities of ESSA which brings together the experimental, thermodynamic and comparative methods, are increased by coupling it with a program dedicated to RNA folding prediction based on constraints management and propagation. The potentialities of ESSA are illustrated by the identification of a possible tertiary motif in the LSU rRNA and the visualization of a pseudoknot in S15 mRNA.

Base Composition↗
PubMed2005

RNA secondary structural alignment with conditional random fields.

MOTIVATION: The computational identification of non-coding RNA regions on the genome is currently receiving much attention. However, it is essentially harder than gene-finding problems for protein-coding regions because non-coding RNA sequences do not have strong statistical signals. Since comparative sequence analysis is effective for non-coding RNA detection, efficient computational methods are expected for structural alignment of RNA sequences. Several methods have been proposed to accomplish the structural alignment tasks for RNA sequences, and we found that one of the most important points is to estimate an accurate score matrix for calculating structural alignments. RESULTS: We propose a novel approach for RNA structural alignment based on conditional random fields (CRFs). Our approach has some specific features compared with previous methods in the sense that the parameters for structural alignment are estimated such that the model can most probably discriminate between correct alignments and incorrect alignments, and has the generalization ability so that a satisfiable score matrix can be obtained even with a small number of sample data without overfitting. Experimental results clearly show that the parameter estimation with CRFs can outperform all the other existing methods for structural alignments of RNA sequences. Furthermore, structural alignment search based on CRFs is more accurate for predicting non-coding RNA regions than the other scoring methods. These experimental results strongly support our discriminative method employing CRFs to estimate the score matrix parameters. AVAILABILITY: The program which is implemented in C++ is available at http://phmmts.dna.bio.keio.ac.jp/ under the GNU public license.

Base Sequence↗
PubMed1998

To knot or not to knot? Examination of 16S ribosomal RNA models.

The presence of topological knots in large RNA structures is highly unlikely given that 1) no RNA structures determined thus far contain topological knots, 2) secondary structure maps for most RNA molecules are knot free, 3) there are no known RNA topoisomerases, and 4) it is difficult to imagine how knots could be formed specifically and uniquely during transcription. Since native RNA structures probably lack topological knots, models of these RNA molecules should be free of knots as well. Therefore, we have examined four existing models for the 30S ribosomal subunit to determine if any of the three domains of the 16S rRNA molecule is knotted. We found that all but one model had at least one knotted domain. We conclude that models of large RNA molecules should be examined for knotting before publication.

Computer Simulation↗
PubMed2006

Comparison of P-RnaPredict and mfold--algorithms for RNA secondary structure prediction.

MOTIVATION: Ribonucleic acid is vital in numerous stages of protein synthesis; it also possesses important functional and structural roles within the cell. The function of an RNA molecule within a particular organic system is principally determined by its structure. The current physical methods available for structure determination are time-consuming and expensive. Hence, computational methods for structure prediction are sought after. The energies involved by the formation of secondary structure elements are significantly greater than those of tertiary elements. Therefore, RNA structure prediction focuses on secondary structure. RESULTS: We present P-RnaPredict, a parallel evolutionary algorithm for RNA secondary structure prediction. The speedup provided by parallelization is investigated with five sequences, and a dramatic improvement in speedup is demonstrated, especially with longer sequences. An evaluation of the performance of P-RnaPredict in terms of prediction accuracy is made through comparison with 10 individual known structures from 3 RNA classes (5S rRNA, Group I intron 16S rRNA and 16S rRNA) and the mfold dynamic programming algorithm. P-RnaPredict is able to predict structures with higher true positive base pair counts and lower false positives than mfold on certain sequences. AVAILABILITY: P-RnaPredict is available for non-commercial usage. Interested parties should contact Kay C. Wiese (wiese@cs.sfu.ca).

Algorithms↗
PubMed2002

Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus.

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core- and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.

Base Sequence↗
PubMed1996

Hammering away at RNA global structure.

A major goal of the study of RNA tertiary structure is an understanding of the rules relating sequence and global conformation. This goal has been furthered during the past year for two important structural elements: yeast tRNAPhe and the self-cleaving hammerhead RNA. In both cases, a combination of solution and crystallographic studies has yielded strongly concordant views of their global conformations.

Base Sequence↗
PubMed1992

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Multiple coding and the evolutionary properties of RNA secondary structure.

This article evaluates evolutionary properties of the transition from RNA primary sequence to RNA secondary structure. It focuses on the restrictions that the conservation of a protein code in an RNA sequence puts on its potential to evolve towards a specific secondary structure. Restricting the mutations to those that do not affect the coding for a protein restricts both the accessibility and the connectivity of the sequence space. The accessibility is restricted because only certain point mutations are allowed. The connectivity is restricted because no insertions and deletions are allowed. Simulating an evolutionary search process for a specific secondary structure shows that (i) the reduction of allowable point mutations allows for adaptation to some large-scale topology, but strongly reduces the possibility of small-scale adaptations, (ii) the abolition of insertions and deletions has very little effect on the results of the search process. During the evolutionary search process for a secondary structure with a specific topology and a high frequency of base-pairing the quasispecies moves into a subspace in which the similarity between secondary structures of neighboring sequences is relatively high. Increased similarity between second structures of neighboring sequences is also found in the Rev responsive element (RRE) in the lentiviruses Caprine arthritis-encephalitis virus and Visna virus. In these viruses a biased nucleotide frequency in the RRE region suggests that selection for the RRE RNA secondary structure affects the amino acid sequence of the env gene. Our results show a variation in the ruggedness of fitness landscapes which are based on a high degree of epistatic interactions. Fitness landscapes play an essential role, not only in biotic evolution, but also in all kinds of optimization processes (Hill Climbing, Simulated Annealing, Genetic Algorithms, etc). Variation in their ruggedness should therefore be taken into account in the analysis of these processes.

Amino Acid Sequence↗
PubMed2005

On a six-dimensional representation of RNA secondary structures.

In this paper, we proposed a 6-D representation of RNA secondary structures. The use of the 6-D representation is illustrated by constructing structure invariants. Comparisons with the similarity/dissimilarity results based on 6-D representation for a set of RNA secondary structures, are considered to illustrate the use of our structure invariants based on the entries in derived sequence matrices restricted to a selected width of a band along the main diagonal.

Base Pairing↗
PubMed1982

The importance of RNA secondary structure in CoIE1 primer formation.

Formation of the RNA primer for CoIE1 DNA replication is inhibited by random substitution of less than one tenth of G residues by I residues during in vitro transcription. Substitution in any one of several regions of the transcript is inhibitory, even in the region more than 400 nucleotides upstream of the origin of DNA replication. The inhibition results from interference with hybrid formation between nascent RNA II (primer transcript) and the template DNA near the replication origin. Association of RNA I with RNA II, which has been known to inhibit primer formation, enhances pausing of transcription of RNA II at a site far downstream of the region where RNA I hybridizes to the transcript. A large deletion in the region which specifies both RNA I and RNA II suppresses primer formation and also enhances pausing of transcription at the same site. These results show that the secondary structure of RNA II during transcription is important for primer formation and that alteration in the structure of the nascent transcript can change transcriptional events far downstream.

Base Sequence↗
PubMed1999

RNA secondary structure and compensatory evolution.

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.

Alcohol Dehydrogenase↗
PubMed2004

Investigation of the Bernoulli model for RNA secondary structures.

Within this paper we investigate the Bernoulli model for random secondary structures of ribonucleic acid (RNA) molecules. Assuming that two random bases can form a hydrogen bond with probability p we prove asymptotic equivalents for the averaged number of hairpins and bulges, the averaged loop length, the expected order, the expected number of secondary structures of size n and order k and further parameters all depending on p. In this way we get an insight into the change of shape of a random structure during the process 1 p -->0. Afterwards we compare the computed parameters for random structures in the Bernoulli model to the corresponding quantities for real existing secondary structures of large subunit rRNA molecules found in the database of Wuyts et al. That is how it becomes possible to identify those parameters which behave (almost) randomly and those which do not and thus should be considered as interesting, e.g., with respect to the biological functions or the algorithmic prediction of RNA secondary structures.

Hydrogen Bonding↗
PubMed1991

RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus.

We have examined the cis-acting RNA packaging signal (psi) from Moloney murine leukemia virus using a combination of chemical and primary sequence analysis techniques. For our chemical analyses, we used dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate as probes for RNA secondary structure. The structural information obtained from these studies was used to constrain computer algorithms for prediction of RNA secondary structure. In addition, we generated and analyzed a phylogenetic comparison of homologous sequences from related retroviruses. From these data, we have developed two models for the RNA secondary structure of the packaging signal psi. Both of these models suggest the presence of secondary structure elements in a region of the psi RNA known to be required for function.

Aldehydes↗
PubMed1997

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Amino Acid Sequence↗