Molecular dynamics simulation of RNA pseudoknot unfolding pathway

Many biological functions of RNA molecules are related to their pseudoknot structures. It is significant for predicting the structure and function of RNA that learning about the stability and the process of RNA pseudoknot folding and unfolding. The structural features of mouse mammary tumor virus (MMTV) RNA pseudoknot in different ion concentration, the unfolding process of the RNA pseudoknot, and the two hairpin helices that constitute the RNA pseudoknot were studied with all atom molecule dynamics simulation method in this paper. We found that the higher cation concentration can cause structure of the RNA molecules more stable, and ions played an indispensable role in keeping the structure of RNA molecules stable; the unfolding process of hairpin structure was corresponding to the antiprocess of its folding process. The main pathway of pseudoknot unfolding was that the inner base pair opened first, and then, the two helices, which formed the RNA pseudoknot opened decussately, while the folding pathway of the RNA pseudoknot was a helix folding after formation of the other helix. Therefore, the unfolding process of RNA pseudoknot is different from the antiprocess of its folding process, and the unfolding process of each helix in the RNA pseudoknot is similar to the hairpin structure’s unfolding process, which means that both are the unzipping process.     

Stable RNA hairpins in 88 coding regions of human mRNA

RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R＝G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.     

Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase

The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules. The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein. These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.     

基于改进适应度函数的RNA结构预测

RNA二级结构预测是生物信息学的重要研究内容。本文提出了一个新的启发式算法进行带假节的RNA结构预测。本文首先通过对RNA序列的若干特征和RNA二级结构进行相关性分析,从中选择跟RNA结构有较大相关性的特征,然后依据遗传算法、综合自由能、被选择茎区的条数以及被选择茎区的平均长度等特征来构造打分函数预测RNA的结构。本文对该方法进行了测试,结果表明本文所采用的从特征分析中得出的打分函数以及通过启发式算法来叠加茎的方法是有效的,在对tRNA以及5SrRNA等序列的预测上相比单纯的自由能最小方法有更高的准确性。并且该方法进一步推广到预测含假结的RNA的二级结构时也有较好的结果。     

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.     

The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data

The classical RNA secondary structure model considers A.U and G.C Watson-Crick as well as G.U wobble base pairs. Here we substitute it for a new one, in which sets of nucleotide cyclic motifs define RNA structures. This model allows us to unify all base pairing energetic contributions in an effective scoring function to tackle the problem of RNA folding. We show how pipelining two computer algorithms based on nucleotide cyclic motifs, MC-Fold and MC-Sym, reproduces a series of experimentally determined RNA three-dimensional structures from the sequence. This demonstrates how crucial the consideration of all base-pairing interactions is in filling the gap between sequence and structure. We use the pipeline to define rules of precursor microRNA folding in double helices, despite the presence of a number of presumed mismatches and bulges, and to propose a new model of the human immunodeficiency virus-1 -1 frame-shifting element.     

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.     

X-ray structure of a DNA hairpin molecule

We have solved the crystal structure of a synthetic DNA hexadecanucleotide of sequence: C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, at 2.1 A resolution, and observed that it adopts a monomeric hairpin configuration with a Z-DNA hexamer stem. In the T4 loop the bases stack with one another and with neighbouring molecules of the crystal, and not with base pairs of their own hexamer stem. Two thymine T10 rings from different molecules stack between the C1-G16 ends of a third and a fourth hairpin helix, in a manner that suggests T-T base 'pairing' and simulates a long, 13-base-pair helix. Although such T-T interactions would not be present in solution, they illustrate a remarkable tendency of thymines for self-association. Purine-purine G-A base pairs are known to exist in the anti-anti conformation with an increase in local helix width; it may be that more serious consideration should be given to the possible existence of pyrimidine-pyrimidine C-T base pairs with decreased local helix width, particularly where several such base pairs occur sequentially.     

The three-dimensional structure of a DNA duplex containing looped-out bases

Unpaired bases in DNA have been assigned a possible role in the mechanism of frameshift mutagenesis in sequences with repeated base pairs. They also occur in quasipalindromic DNA sequences, which have been implicated in mutagenesis where there are no repeated base pairs, through the formation of single-stranded hairpin loops. The conformation of unpaired bases in DNA has been the subject of numerous thermodynamic as well as high resolution NMR (nuclear magnetic resonance) studies (reviewed in ref. 4). The NMR studies in solution have shown that the duplex of the tridecamer DNA fragment d(CGCAGAATTCGCG) remains intact, and that the unpaired adenosines are stacked into the duplex. Having crystallized this oligonucleotide and determined its structure, we find its conformation in the crystal is close to that of a B-DNA duplex, with the two additional adenosines looped out from the double helix and causing little disruption of the rest of the structure.     

Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies

Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects-features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements and have already provided information on the folding free energy of a RNA hairpin. Here we show that the Crooks fluctuation theorem can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.     

一个预测RNA二级结构的回溯算法

本文讨论了一个预测RNA二级结构的回溯算法。该算法根据极大基配对的原则按字典顺序产生所有可能的二级结构。它的时间复杂性是O(n~2),空间复杂性是O(n)。     

Prediction for RNA planar pseudoknots

Based on m-stems and semi-extensible structure, a model is presented to represent RNA planar pseudoknots, and corresponding dynamic programming algorithm is designed and implemented to predict arbitrary planar pseudoknots and simple non-planar pseudoknots with O(n4) time and O(n3) space. The algorithm folds total 245 sequences in the Pseudobase database, and the test results indicate that the algorithm has good accuracy, sensitivity and specificity.     

The topological configuration and conformational analysis of mRNA in translation

The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.     

RNA二级结构自动绘图系统的构建

给出了带假结的RNA二级结构自动绘画系统以及自由能的处理．用软件实现此方法可以把以碱基匹配表示的RNA分子二级结构自动转化为图形表示．     

Structure of yeast Argonaute with guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 ? crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.     

A peptide model of a protein folding intermediate

It is difficult to determine the structures of protein folding intermediates because folding is a highly cooperative process. A disulphide-bonded peptide pair, designed to mimic the first crucial intermediate in the folding of bovine pancreatic trypsin inhibitor, contains secondary and tertiary structure similar to that found in the native protein. Peptide models like this circumvent the problem of cooperativity and permit characterization of structures of folding intermediates.     

Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.