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PubMed1995

Mutational analysis of the 3'-terminal extra-cistronic region of poliovirus RNA: secondary structure is not the only requirement for minus strand RNA replication.

A series of mutations were introduced in the 3'-terminal untranslated region (3'-UTR) of full-length infectious poliovirus cDNA clones, and following transfection of COS-1 cells the ability of these constructs to generate viable viral particles and/or to support viral RNA synthesis was assayed. Substitution of the 3'-UTR of poliovirus RNA with the equivalent sequences of HAV RNA abrogated viral RNA replication, whereas the introduction of extended 'foreign' sequences between the open reading frame and the 3'-UTR was well tolerated. Point mutation that either destabilized the stem-and-loop structure or altered the sequence of the loop in domain 'Y' (nomenclature as per Pilipenko et al., [Nuclei Acids Res. 20 (1992) 1739-1745]) abolished both the infectivity and viral RNA synthesis. These were not restored by compensatory mutation that reconstructed the native secondary structure of this domain, suggesting that the secondary/tertiary folding of the 3'-UTR is not the only determinant for template recognition at initiation of RNA synthesis, but rather that a specific primary sequence is indeed required.

Animals↗

PubMed1994

RNA tertiary structure of the HIV RRE domain II containing non-Watson-Crick base pairs GG and GA: molecular modeling studies.

We have used molecular modeling techniques to model the RNA tertiary structure of the viral RNA element (referred to as domain II of Rev responsive element, RRE) bound by the Rev protein of HIV. In this study, the initial three-dimensional model was built from its established RNA secondary structure, including three non-Watson-Crick G:G, G:A and G:U base pairs. Molecular dynamics (MD) simulations were performed with hydrated or unhydrated sodium ions. Our results indicate that the non-Watson-Crick base pairs in the simulation with unhydrated sodium ions and water are more stable than those with hydrated sodium ions only. The RNA can maintain its compact double helical structure throughout the course of the MD simulations with water and unhydrated sodium ions, although the non-Watson-Crick base pairs and two bulge loops show much more flexibility and conformational distortion than the classical RNA helical region. The distinct distortion of the sugar-phosphate backbone significantly widens the RNA major groove so that the major groove is readily accessible for hydrogen bonding by specific Rev binding. This model emphasizes the importance of specific hydrogen bonding in the stabilization of the three-dimensional structure of the HIV Rev core binding element, not only between the nucleotide bases, but also among the ribose hydroxyls, phosphate anionic oxygens, base oxygens and nitrogens, and bridging water molecules. Moreover, our results suggest that sodium ions play an important role in the formation of base pairs G:G and G:A of the RRE by a manner similar to the arginine of the Rev-RRE complex.

Adenine↗

PubMedDate not supplied

Design and analysis of RNA structure-specific agents as potential antivirals.

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants > 10(7)M-1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

Antiviral Agents↗

PubMed2002

A compact RNA tertiary structure contains a buried backbone-K+ complex.

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Base Sequence↗

PubMed1996

The C-terminal domain revealed in the structure of RNA polymerase II.

The location of the CTD in the structure of RNA polymerase II has been determined by electron crystallography at 16 A resolution. Difference maps between wild-type enzyme and that lacking the CTD, or with an antibody fragment bound in place of the CTD, disclose the site of attachment of the CTD to the polymerase. Appropriate display of the polymerase structure reveals the CTD as an element projecting from this site of attachment into solution. A low relative density and large volume of this element identify the CTD as a conformationally mobile region.

Amino Acid Sequence↗

PubMed2006

A method for finding optimal rna secondary structures using a new entropy model (vsfold).

We are developing a program to calculate optimal RNA secondary structures. The model uses di-nucleotide pairing energies as with most traditional approaches. However, for long-range entropy interactions, the approach uses an entropy-loss model based on the accumulated sum of the entropy of bonding between each base-pair weighted inversely by the correlation of the RNA sequence (the Kuhn length). Stiff RNA forms very different structures from flexible RNA. The results demonstrate that the long-range folding is largely governed by this entropy and the Kuhn length.

Entropy↗

PubMed2004

An iterated loop matching approach to the prediction of RNA secondary structures with pseudoknots.

MOTIVATION: Pseudoknots have generally been excluded from the prediction of RNA secondary structures due to its difficulty in modeling. Although, several dynamic programming algorithms exist for the prediction of pseudoknots using thermodynamic approaches, they are neither reliable nor efficient. On the other hand, comparative methods are more reliable, but are often done in an ad hoc manner and require expert intervention. Maximum weighted matching, an algorithm for pseudoknot prediction with comparative analysis, suffers from low-prediction accuracy in many cases. RESULTS: Here we present an algorithm, iterated loop matching, for reliably and efficiently predicting RNA secondary structures including pseudoknots. The method can utilize either thermodynamic or comparative information or both, thus is able to predict pseudoknots for both aligned and individual sequences. We have tested the algorithm on a number of RNA families. Using 8-12 homologous sequences, the algorithm correctly identifies more than 90% of base-pairs for short sequences and 80% overall. It correctly predicts nearly all pseudoknots and produces very few spurious base-pairs for sequences without pseudoknots. Comparisons show that our algorithm is both more sensitive and more specific than the maximum weighted matching method. In addition, our algorithm has high-prediction accuracy on individual sequences, comparable with the PKNOTS algorithm, while using much less computational resources. AVAILABILITY: The program has been implemented in ANSI C and is freely available for academic use at http://www.cse.wustl.edu/~zhang/projects/rna/ilm/ SUPPLEMENTARY INFORMATION: http://www.cse.wustl.edu/~zhang/projects/rna/ilm/

Algorithms↗

PubMed1999

Fast evaluation of internal loops in RNA secondary structure prediction.

MOTIVATION: Though not as abundant in known biological processes as proteins, RNA molecules serve as more than mere intermediaries between DNA and proteins. Research in the last 15 years demonstrates that RNA molecules serve in many roles, including catalysis. Furthermore, RNA secondary structure prediction based on free energy rules for stacking and loop formation remains one of the few major breakthroughs in the field of structure prediction, as minimum free energy structures and related quantities can be computed with full mathematical rigor. However, with the current energy parameters, the algorithms used hitherto suffer the disadvantage of either employing heuristics that risk (though highly unlikely) missing the optimal structure or becoming prohibitively time consuming for moderate to large sequences. RESULTS: We present a new method to evaluate internal loops utilizing currently used energy rules. This method reduces the time complexity of this part of the structure prediction from O(n4) to O(n3), thus reducing the overall complexity to O(n3). Even when the size of evaluated internal loops is bounded by k (a commonly used heuristic), the method presented has a competitive edge by reducing the time complexity of internal loop evaluation from O(k2n2) to O(kn2). The method also applies to the calculation of the equilibrium partition function. AVAILABILITY: Source code for an RNA secondary structure prediction program implementing this method is available at ftp://www.ibc.wustl.edu/pub/zuker/zuker .tar.Z

Algorithms↗

PubMed2003

Predicting RNA secondary structures with arbitrary pseudoknots by maximizing the number of stacking pairs.

The paper investigates the computational problem of predicting RNA secondary structures. The general belief is that allowing pseudoknots makes the problem hard. Existing polynomial-time algorithms are heuristic algorithms with no performance guarantee and can handle only limited types of pseudoknots. In this paper, we initiate the study of predicting RNA secondary structures with a maximum number of stacking pairs while allowing arbitrary pseudoknots. We obtain two approximation algorithms with worst-case approximation ratios of 1/2 and 1/3 for planar and general secondary structures, respectively. For an RNA sequence of n bases, the approximation algorithm for planar secondary structures runs in O(n(3)) time while that for the general case runs in linear time. Furthermore, we prove that allowing pseudoknots makes it NP-hard to maximize the number of stacking pairs in a planar secondary structure. This result is in contrast with the recent NP-hard results on psuedoknots which are based on optimizing some general and complicated energy functions.

Algorithms↗

PubMed1993

Modeling the three-dimensional structure of RNA using discrete nucleotide conformational sets.

The flexibility about seven torsion angles in nucleotides constitutes a severe obstacle to computer modeling of RNA. The computational feasibility of RNA conformational searches can be enhanced by assigning to each nucleotide a set of discrete conformations. In this work, four types of discrete conformational sets for the atomic representation of nucleotide structures were defined and evaluated. These sets, comprising between 10 and 30 conformations, were tested for their ability to reproduce known RNA structures and to generate structures responding to new specifications. Conformational searches were performed with the MC-SYM program, which allows for the generation of all structures satisfying a predetermined set of three-dimensional constraints in a given discrete space. Results with known hairpin loop structures show that root-mean-square deviations of about 1.5 A for backbone atoms and about 2.0 A for all atoms between the modeled and X-ray crystal structures can be expected. The conformational set that gives the most faithful representation of test structures is based on the classification of nucleotide conformations derived from a structural database. Representative conformations are selected from each class that adequately sample variations in backbone direction, sugar pucker and base orientation. With this conformational set, most of the important features of test hairpin structures are reproduced with fidelity, indicating that biologically useful models can be constructed from the combination of discrete nucleotide conformations and an algorithm that rapidly and systematically scans the pre-defined conformational space.

Anticodon↗

PubMed1995

Symmetry and structure of RNA and DNA triple helices.

Despite wide interest in nucleic acid triple helices, there has been no stereochemically satisfactory structure of an RNA triple helix in atomic detail. AN RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244, pp. 99-101; S. Arnott, P. J. Bond, E. Selsing, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3, pp. 2459-2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new model for an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3-11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from the introduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455-462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu, H. T. Miles, K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1, pp. 11-12]. The above triple helices have Watson-Crick-Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16, pp. 907-916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson-Crick-reverse Hoogsteen, Donohue [J. Donohue (1953) Proceedings of the National Academy of Science USA, Vol. 39, pp. 470-475] (reverse Watson-Crick)-Hoogsteen, and Donohue-reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain.

DNA↗

PubMed1993

A toolkit for creating three-dimensional RNA pencil models.

The advantages of nucleotide-resolution models over atomic-resolution and cylinder models of large RNA structure are discussed, and a toolkit of RNA substructures for use in building 'pencil' models is described. Prefabricated elements from the toolkit can be used to quickly assemble complex RNA structure models for the visualization of known RNA structures, or for exploring potential tertiary structure configurations based on secondary structure and other information.

Computer-Aided Design↗

PubMed1989

RNA secondary structures: comparison and determination of frequently recurring substructures by consensus.

A method for assessing the preserved stem-loops of RNA secondary structures is presented. Frequently recurring helical stems in a set of secondary structures resulting from the simulated folding process of a given RNA are assessed and consensus structural motifs can then be selected to construct a secondary structure of the RNA. Alternatively, it can be applied to a series of 'optimal' and 'suboptimal' secondary structures computed using the dynamic program developed by Williams and Tinoco. To demonstrate the power and the usefulness of the program we give examples of this procedure.

Algorithms↗

PubMed1999

Repression of IS200 transposase synthesis by RNA secondary structures.

The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis approximately 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200.

Base Pairing↗

PubMed1998

RNA secondary structure: an important cis-element in rat calcitonin/CGRP pre-messenger RNA splicing.

The calcitonin/CGRP gene pre-mRNA is alternatively spliced in a tissue-specific manner resulting in the formation of calcitonin mRNA in thyroid C-cells and CGRP mRNA in neurons. Computer analysis of the RNA containing the 3' splice acceptor of the calcitonin-specific exon 4 predicts that this region has the potential to form a thermodynamically stable stem-loop. Data from CD spectroscopy and solution phase structure probing with single-strand specific and double-strand specific RNases indicates that RNA in this region is substantially double stranded. In vitro splicing of chimeric human beta-globin/calcitonin transcripts in HeLa nuclear extract was inhibited by base changes predicted to destabilize the stem, while compensatory base changes resulted in splicing at 50% of wild-type levels. Changing the residue opposite the AG dinucleotide adenosine in the stem from G to U, allowing the formation of an A-U basepair, abolished usage of this splice acceptor in vitro. These results indicate that a thermodynamically stable RNA stem-loop forms in vitro at the 3' splice acceptor of exon 4 of the calcitonin/CGRP gene transcript. This RNA secondary structure acts as a novel cis-element involved in proper splice site selection.

Alternative Splicing↗

PubMed1987

Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2.

The lysis gene of the RNA bacteriophage MS2 is not expressed unless translation of the overlapping coat gene takes place. To understand the molecular basis for this translational coupling the RNA secondary structure around the lysis gene start was analyzed with structure-specific enzymes and chemicals. The existence of a hairpin between nucleotides 1636 and 1707 is in agreement with the structural mapping data and also with the conservation of base-pairing in the related M12 phage. In this hairpin, the G residues in the Shine and Dalgarno region and start codon are inaccessible to RNase T1, which is consistent with the fact that ribosomal access to the lysis gene is blocked when there is no coat gene translation. Deletions or point mutations that are predicted to destabilize the hairpin give rise to lysis protein synthesis that is independent of coat gene translation. Base substitutions that are not expected to weaken the helix do not lead to independent lysis gene expression. Finally, nucleotide changes that strengthen the hairpin lead neither to uncoupled nor to coupled synthesis of the lysis protein. Structural analysis of mutant MS2 RNA shows that small changes in the stability of the secondary structure lead to substantial differences in translation initiation. The function of the hairpin structure in coupling lysis gene to coat gene translation requires that its stability is kept within narrow limits.

Bacteriophages↗

PubMed2000

Glassy transition in a disordered model for the RNA secondary structure.

We numerically study a disordered model for the RNA secondary structure and we find that it undergoes a phase transition, with a breaking of the replica symmetry in the low temperature region (as in spin glasses). Our results are based on the exact evaluation of the partition function.

Models, Chemical↗

PubMed1988

An RNA secondary structure workbench.

A multiple approach to the study of RNA secondary structure is described which provides for the independent drawing of structures using base-pairing lists, for the generation of local structures in the form of hairpins, and for the generation of global structures by both Monte Carlo and dynamic programming methodologies. User-adjustable parameters provide for limiting the size of hairpin loops, bulges and inner loops, and constraints can be imposed relative to position-dependent base pairing.

Algorithms↗