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PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗
PubMed2000

Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus and Pestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T(1)-probing studies, demonstrating the importance of correct RNA folding to IRES function.

5' Untranslated Regions↗
PubMed1998

Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis.

The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5' end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963-4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not negative-strand RNA structure, was important for the biological function of the region. (iii) The biological function had a sequence-specific requirement. We discuss possible mechanisms by which the internal cis-acting signal drives MHV positive-strand DI RNA synthesis.

Animals↗
PubMed2002

Immobilized small deoxyribozyme to distinguish RNA secondary structures.

The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.

Base Sequence↗
PubMed1979

Studies on the specificity and clinical correlation of antiribosomal antibodies in systemic lupus erythematosus sera.

The specificity of antibodies reactive with cytoplasmic ribosomes detected in systemic lupus erythematosus (SLE) sera was studied by a radioimmunoassay procedure with 3H labeled HeLa cell polysomes. The antibodies were directed primarily against RNA or a ribosomal RNA-protein complex (rRNP). Anti-RNA antibodies exhibited comparable reactivity with ribosomal RNA, nuclear RNA, structural RNA, and a slightly lower degree of reactivity with low molecular weight RNA. Anti-rRNP antibodies may be detected by precipitation in agar gel and appear to require both the RNA and protein moieties of ribosomes for reactivity. SLE sera containing only anti-rRNP antibodies are limited to patients with active disease. Sera from patients with both active and inactive SLE contain either anti-RNA or a mixture of anti-RNA and anti-rRNP antibodies. The possible participation of anti-ribosomal antibodies in immune complex formation is discussed.

Antibodies, Antinuclear↗
PubMed1999

The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.

Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method.

Base Sequence↗
PubMed2004

Evaluation of several lightweight stochastic context-free grammars for RNA secondary structure prediction.

BACKGROUND: RNA secondary structure prediction methods based on probabilistic modeling can be developed using stochastic context-free grammars (SCFGs). Such methods can readily combine different sources of information that can be expressed probabilistically, such as an evolutionary model of comparative RNA sequence analysis and a biophysical model of structure plausibility. However, the number of free parameters in an integrated model for consensus RNA structure prediction can become untenable if the underlying SCFG design is too complex. Thus a key question is, what small, simple SCFG designs perform best for RNA secondary structure prediction? RESULTS: Nine different small SCFGs were implemented to explore the tradeoffs between model complexity and prediction accuracy. Each model was tested for single sequence structure prediction accuracy on a benchmark set of RNA secondary structures. CONCLUSIONS: Four SCFG designs had prediction accuracies near the performance of current energy minimization programs. One of these designs, introduced by Knudsen and Hein in their PFOLD algorithm, has only 21 free parameters and is significantly simpler than the others.

Computational Biology↗
PubMed2004

Single molecule studies of RNA secondary structure: AFM of TYMV viral RNA.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

Image Processing, Computer-Assisted↗
PubMed1996

A graph-topological approach to recognition of pattern and similarity in RNA secondary structures.

Secondary and tertiary RNA structures play an important role in many biological processes. Therefore the necessity arises to find similar higher-order structures for different but functionally homologous RNA sequences. We propose here a graph-topological approach to the problem, which shows two main features: simplified graph representation which allows the recognition of similarity of RNA secondary structures with the same branching look despite minor differences. This allows comparison among foldings from different sequences, and "pruning" of the secondary structures not shared by all the sequences since the early stages of the search. (b) The graph representation is encoded by the Randić topological index, and the search for the folding similarity is reduced to checking the identity of single numbers. These characteristics make this approach significantly different, less depending on empirical criteria, and less computationally heavy then previous methods, where the folding consensus has been measured by an alignment procedure or correlation of strings representing the secondary structures. Some U2 snRNA and viroid sequences are studied by this approach, which is imbedded in our previous search method based on genetic algorithms.

Algorithms↗
PubMed1999

RNA movies: visualizing RNA secondary structure spaces.

MOTIVATION: RNA Movies is a system for the visualization of RNA secondary structure spaces. Its input is a script consisting of primary and secondary structure information. From this script, the system fully automatically generates animated graphical structure representations. In this way, it creates the impression of an RNA molecule exploring its own two-dimensional structure space. RESULTS: RNA Movies has been used to generate animations of a switching structure in the spliced leader RNA of Leptomonas collosoma and sequential foldings of potato spindle tuber viroid transcripts. AVAILABILITY: Demonstrations of the animations mentioned in this paper can be viewed on our Bioinformatics web server under the following address: http://BiBiServ.TechFak.Uni-Bielefeld. DE/rnamovies/. The RNA Movies software is available upon request from the authors.

Algorithms↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗
PubMed1994

Structure of a mispaired RNA double helix at 1.6-A resolution and implications for the prediction of RNA secondary structure.

The nonamer r(GCUUCGGC)dBrU, where dBrU is 5-bromo-2'-deoxyuridine, contains the tetraloop sequence UUCG. It crystallizes in the presence of Rh(NH3)6Cl3. In solution the oligomer is expected to form a hairpin loop but the x-ray structure analysis, to a resolution of 1.6 A, indicates an eight-base-pair A-RNA duplex containing a central block of two G.U and two C.U pairs. Self-pairs which approximate to Watson-Crick geometry are also formed in the extended crystal structure between symmetry-related BrU residues and are part of infinite double-helical stacks. The G.U pair is a wobble base pair analogous to the G.T pair found in DNA fragments. The C.U mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit of the base pair in the RNA helix. The BrU.BrU pair is held by two hydrogen bonds in an orientation which is compatible with duplex geometry. The structure observed within the crystal has some parallels with the structure of globular RNAs, and the presence of stable, noncanonical base pairs has implications for the prediction of RNA secondary structure.

Base Composition↗
PubMed2004

RDfolder: a web server for prediction of RNA secondary structure.

Prediction of RNA secondary structure is important in the functional analysis of RNA molecules. The RDfolder web server described in this paper provides two methods for prediction of RNA secondary structure: random stacking of helical regions and helical regions distribution. The random stacking method predicts secondary structure by Monte Carlo simulations. The method of helical regions distribution predicts secondary structure based on the helices that appear most frequently in the set of structures, which are generated by the random stacking method. The RDfolder web server can be accessed at http://rna.cbi.pku.edu.cn.

Internet↗
PubMed1998

Insights into Escherichia coli RNA polymerase structure from a combination of x-ray and electron crystallography.

Our goal is to understand the mechanism of transcription and its regulation. Determining structures of RNA polymerase and transcription complexes is an essential step. Because of their large size and complexity, determination of these structures will require a combination of electron microscopy, biophysical methods, and biochemical methods to identify functionally and structurally relevant subassemblies and domains and x-ray crystallography to determine high-resolution structures of RNA polymerase components and accessory factors. We recently solved the 2.5-A crystal structure of the Escherichia coli RNA polymerase alpha subunit N-terminal domain, which is the first high-resolution structure of a core component required for RNA polymerase assembly and basal transcription. This structure, combined with a new 19-A resolution structure determined by cryo-electron microscopy of helical crystals of E. coli core RNAP embedded in vitreous ice, leads to a model for the organization of the RNAP subunits.

Crystallography, X-Ray↗
PubMed2003

Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.

Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by </=2-fold. The absence of effects on k(dock), both from atomic modifications and global perturbations, strongly suggests that the transition state for docking is early and does not closely resemble the docked state. These results, the slow rate of docking of 3s(-1), and the observation that a modification that is expected to increase the degrees of freedom between the P1 duplex and the ribozyme core accelerates docking, suggest a model in which a kinetic trap(s) slows docking substantially. Nonetheless, urea does not increase k(dock), suggesting that there is little change in the exposed surface area between the trapped, undocked state and the transition state. The findings highlight that urea and temperature dependencies can be inadequate to diagnose the presence of kinetic traps in a folding process. The results described here, combined with previous work, provide an in-depth view of an RNA tertiary structure formation event and suggest that large, highly structured RNAs may have local regions that are misordered.

Animals↗
PubMed1984

Equilibria in 5-S ribosomal RNA secondary structure. Bulges and interior loops in 5-S RNA secondary structure may serve as articulations for a flexible molecule.

The basic assumption in this paper is that the secondary structure of a 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In eubacterial 5-S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure.

Animals↗
PubMed2004

Evolutionary rate variation and RNA secondary structure prediction.

Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type of approach. Determining these rates can be hard to do reliably without a large and accurate initial alignment, which ideally also has structural annotation. Hence, one must often apply rates extracted from other RNA families with trusted alignments and structures. Here, we investigate this problem by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions. In addition we obtained an alignment of the 5' HIV-1 region that is more consistent with the structure than that currently in the database. We added randomized noise to the original values of the rates to investigate the stability of predictions to rate matrix deviations. We find that changes within a fairly large range still produce reliable predictions and conclude that using rates from a limited set of RNA sequences is valid over a broader range of sequences.

Algorithms↗