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At least 1,387 records · Page 77Linked to original sources
PubMed1990

Metabolic complexity in the RNA world and implications for the origin of protein synthesis.

A model is presented for the evolution of metabolism and protein synthesis in a primitive, acellular RNA world. It has been argued previously that the ability to perform metabolic functions logically must have preceded the evolution of a message-dependent protein synthetic machinery and that considerable metabolic complexity was achieved by ribo-organisms (i.e., organisms in which both genome and enzymes are comprised of RNA). The model proposed here offers a mechanism to account for the gradual development of sophisticated metabolic activities by ribo-organisms and explains how such metabolic complexity would lead subsequently to the synthesis of genetically encoded polypeptides. RNA structures ancestral to modern ribosomes, here termed metabolosomes, are proposed to have functioned as organizing centers that coordinated, using base-pairing interactions, the order and nature of adaptor-mounted substrate/catalyst interactions in primitive metabolic pathways. In this way an ancient genetic code for metabolism is envisaged to have predated the specialized modern genetic code for protein synthesis. Thus, encoded amino acids initially would have been used, in conjunction with other encoded metabolites, as building blocks for biosynthetic pathways, a role that they retain in the metabolism of contemporary organisms. At a later stage the encoded amino acids would have been condensed together on similar RNA metabolosome structures to form the first genetically determined, and therefore biologically meaningful, polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic Code↗
PubMed1995

Enediyne-mediated cleavage of RNA.

RNA cleavage by enediyne anticancer antibiotics was shown to occur with no apparent sequence selectivity, but RNA structure appears to be important in those substrates where cleavage was observed. Neocarzinostatin (NCS) cleaved a wider variety of RNA substrates than either esperamicin (ESP) or calicheamicin (CAL), and dynemicin (DYN) has yet to cleave any RNA substrate tried. NCS, ESP, and CAL were all observed to cleave RNA substrates near the 5'-end, and all three compounds exhibited cleavage in single-stranded loop regions of the RNA substrates. NCS required no thiol for activation and subsequent cleavage, but ESP and CAL required addition of thiol, as expected, for cleavage to occur. An RNA hairpin substrate containing a UCCU sequence, equivalent to the TCCT sequence preferred by CAL in double-stranded DNA substrates, was cleaved by CAL, but no retention of selectivity for the UCCU site was retained by CAL in this RNA substrate. This study confirms an earlier observation that RNA is a substrate for enediyne cleavage, and indicates that nucleic acid cleaving compounds such as the enediynes could be useful probes of RNA three-dimensional structure.

Aminoglycosides↗
PubMed2000

Simulation of the structure and dynamics of nonhelical RNA motifs.

Computer simulation methods are increasingly being used to study possible conformations and dynamics of structural motifs in RNA. Recent results of molecular dynamics simulations and continum solvent studies of RNA structures and RNA-ligand complexes show promising agreement with experimental data. Combined with the ongoing progress in the experimental characterization of RNA structure and thermodynamics, these computational approaches can help to better understand the mechanism of RNA structure formation and the binding of ligands.

Animals↗
PubMed2005

Extracting stacking interaction parameters for RNA from the data set of native structures.

A crucial step in the determination of the three-dimensional native structures of RNA is the prediction of their secondary structures, which are stable independent of the tertiary fold. Accurate prediction of the secondary structure requires context-dependent estimates of the interaction parameters. We have exploited the growing database of natively folded RNA structures in the Protein Data Bank (PDB) to obtain stacking interaction parameters using a knowledge-based approach. Remarkably, the calculated values of the resulting statistical potentials (SPs) are in excellent agreement with the parameters determined using measurements in small oligonucleotides. We validate the SPs by predicting 74% of the base-pairs in a dataset of structures using the ViennaRNA package. Interestingly, this number is similar to that obtained using the measured thermodynamic parameters. We also tested the efficacy of the SP in predicting secondary structure by using gapless threading, which we advocate as an alternative method for rapidly predicting RNA structures. For RNA molecules with less than 700 nucleotides, about 70% of the native base-pairs are correctly predicted. As a further validation of the SPs we calculated Z-scores, which measure the relative stability of the native state with respect to a manifold of higher free energy states. The computed Z-scores agree with estimates made using calorimetric measurements for a few RNA molecules. Structural analysis was used to rationalize the success and failures of SP and experimentally determined parameters. First, from the near perfect linear relationship between the number of native base-pairs and sequence length, we show that nearly 46% of nucleotides are not in stacks. Second, by analyzing the suboptimal structures that are generated in gapless threading we show that the SPs and experimentally determined parameters are most successful in predicting stacks that end in hairpins. These results show that further improvement in secondary structure prediction requires reliable estimates of interaction parameters for loops, bulges, and stacks that do not end in hairpins.

Base Pairing↗
PubMedDate not supplied

[Study of the structural organization of RNA from phage MS2 using fluorescent dyes].

The spatial organization of phage MS2 RNA by binding to ethidium bromide (EtBr) and acridine orange (AO) to RNA was studied. The analyses of dye interaction by spectrophotometric and fluorometric methods have demonstrated that only about a half of 65-70% nucleotides of double-stranded segments can interact with AO and EtBr. On the other hand all the single-stranded segments appear to be accessible to AO binding. These interactions did not practically change when ionic strength (0.01-0.3), Mg2+ and Zn2+ concentrations (10(-3) M) or pH (4.7-7.4) varied. The data permit to suppose that phage MS2 RNA has a very stable tertiary structure which makes part of double-stranded segments unaccessible to inter calating dyes. Taking these and other facts into consideration we suppose that double-stranded segments play an important role in stabilization of the RNA tertiary structure. One of the most possible structure is a compact "rod-like" intramolecular aggregate of double-stranded hairpin-like segments of RNA with parallel orientation.

Coliphages↗
PubMed2003

Near native structure in an RNA collapsed state.

Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors. Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood. We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant. RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core. Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent. Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state. Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core. The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently.

Azides↗
PubMed1995

Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA.

The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infection, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is known about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNAI, a highly structured RNA that binds to the kinase. VA RNA contains three chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNAI. DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the central domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain. A similar change is induced by magnesium ions alone. Footprinting and interference binding assays using base-specific chemical probes suggest that the protein does not make major contacts with RNA bases. On the other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove of the apical stem and a weaker interaction in the central domain. A truncated form of DAI, p20, containing only the RNA binding domain, gives a similar protection pattern in the apical stem but protects the central domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of VA RNA, and that other regions of the protein contribute to interactions with the central domain.

Adenoviruses, Human↗
PubMed2001

Design of RNA-binding proteins and ligands.

The rapidly expanding database of RNA structures and protein complexes is beginning to lead to the successful design of specific RNA-binding molecules. Recent combinatorial and structure-based approaches have utilized known nucleic-acid-binding scaffolds from both proteins and small molecules to display a relatively small set of functional groups often used in protein--RNA recognition. Several studies have shown that the tethering of multiple binding modules can enhance RNA-binding affinity and specificity, a strategy also commonly used in DNA recognition.

Animals↗
PubMed2006

Structural basis for non-catalytic and catalytic activities of ribonuclease III.

Ribonuclease III (RNase III) represents a highly conserved family of double-stranded (ds) RNA-specific endoribonucleases, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. Bacterial RNase III, containing an endonuclease domain followed by a dsRNA-binding domain, is the most extensively studied member of the family. It can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves dsRNA or as a binding protein that binds but does not cleave dsRNA. The available biochemical and structural data support the existence of two distinct forms of the RNase III-dsRNA complex which reflect the dual activities of the protein. The information revealed by the structures of bacterial RNase III provides insight into the mechanism of dsRNA processing by all members of the family.

Animals↗
PubMed1999

Development of a system to classify 3D structural character of RNA.

We are developing a computational system to extract structural character of RNA. We made a program that classifies conformers by recognizing the hydrogen bonding patterns. The program was applied to a set of 279 conformers and they were classified into about 40 groups. The system is expected to be useful for searching structural motifs of RNA and classifying large number of generated conformers in structural modeling process.

Base Pairing↗
PubMed2003

Exploring the repertoire of RNA secondary motifs using graph theory; implications for RNA design.

Understanding the structural repertoire of RNA is crucial for RNA genomics research. Yet current methods for finding novel RNAs are limited to small or known RNA families. To expand known RNA structural motifs, we develop a two-dimensional graphical representation approach for describing and estimating the size of RNA's secondary structural repertoire, including naturally occurring and other possible RNA motifs. We employ tree graphs to describe RNA tree motifs and more general (dual) graphs to describe both RNA tree and pseudoknot motifs. Our estimates of RNA's structural space are vastly smaller than the nucleotide sequence space, suggesting a new avenue for finding novel RNAs. Specifically our survey shows that known RNA trees and pseudoknots represent only a small subset of all possible motifs, implying that some of the 'missing' motifs may represent novel RNAs. To help pinpoint RNA-like motifs, we show that the motifs of existing functional RNAs are clustered in a narrow range of topological characteristics. We also illustrate the applications of our approach to the design of novel RNAs and automated comparison of RNA structures; we report several occurrences of RNA motifs within larger RNAs. Thus, our graph theory approach to RNA structures has implications for RNA genomics, structure analysis and design.

Algorithms↗
PubMed2006

The paradoxical behavior of a highly structured misfolded intermediate in RNA folding.

Like many structured RNAs, the Tetrahymena group I ribozyme is prone to misfolding. Here we probe a long-lived misfolded species, referred to as M, and uncover paradoxical aspects of its structure and folding. Previous work indicated that a non-native local secondary structure, termed alt P3, led to formation of M during folding in vitro. Surprisingly, hydroxyl radical footprinting, fluorescence measurements with site-specifically incorporated 2-aminopurine, and functional assays indicate that the native P3, not alt P3, is present in the M state. The paradoxical behavior of alt P3 presumably arises because alt P3 biases folding toward M, but, after commitment to this folding pathway and before formation of M, alt P3 is replaced by P3. Further, structural and functional probes demonstrate that the misfolded ribozyme contains extensive native structure, with only local differences between the two states, and the misfolded structure even possesses partial catalytic activity. Despite the similarity of these structures, re-folding of M to the native state is very slow and is strongly accelerated by urea, Na+, and increased temperature and strongly impeded by Mg2+ and the presence of native peripheral contacts. The paradoxical observations of extensive native structure within the misfolded species but slow conversion of this species to the native state are readily reconciled by a model in which the misfolded state is a topological isomer of the native state, and computational results support the feasibility of this model. We speculate that the complex topology of RNA secondary structures and the inherent rigidity of RNA helices render kinetic traps due to topological isomers considerably more common for RNA than for proteins.

Animals↗
PubMed1993

Chemical probing of tDNAPhe with transition metal complexes: a structural comparison of RNA and DNA.

The three-dimensional folding of tDNAPhe has been examined and compared to native tRNAPhe using a series of shape-selective transition metal complexes as chemical probes of nucleic acid structure. Rh(phen)2 phi 3+ (phen = phenanthroline, phi = 9,10-phenanthrenequinonediimine), which targets sites of tertiary interaction in tRNAPhe, cleaves specifically at similar sites on tDNAPhe. However, this rhodium complex also targets 5'-pyr-pyr-pur-3' sites within the acceptor and anticodon stems of tDNA;Rh(phen)2 phi 3+ generally targets 5'-pyr-pyr-pur-3' sites in B-form duplex DNA. On tRNAPhe, Rh(DIP)3(3+) (DIP = 4,7-diphenyl-1,10-phenanthroline) specifically cleaves C70, which neighbors a GU mismatch, and targets psi 55 as well, within the hydrophobic region of tRNAPhe. On tDNAPhe no specific cleavage by Rh(DIP)3(3+) is observed. The cleavage studies, taken together, indicate that globally the tertiary folding of tDNAPhe resembles that of tRNAPhe. However, the double helical regions of the DNA analog differ from tRNAPhe, likely in adopting a more B-like conformation. As a consequence, the GT mismatch within the acceptor stem of tDNA does not present the same recognition elements as in tRNA, and the GT mismatch is no longer recognized by the shape-selective rhodium complex. The present work underscores the utility of applying DNA analogs to studies of RNA structure and function, since the general folding characteristics of the two polymers are likely to be similar. However, structural probing with transition metal complexes offers a valuable companion to such experiments, since the shape-selective probes, with sensitivity, may be used to delineate locally on the polymer those regions which may differ in structure.

DNA↗
PubMed2005

The double-stranded RNA-binding motif, a versatile macromolecular docking platform.

The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.

Amino Acid Sequence↗
PubMedDate not supplied

De novo design of peptides with L-alpha-nucleobase amino acids and their binding properties to the P22 boxB RNA and its mutants.

A method to design novel molecules that specifically recognize a structured RNA would be a promising tool for the development of drugs or probes targeting RNA. In this study, the de novo design of the alpha-helical peptides having L-alpha-amino acids with nucleobases (nucleobase amino acids, NBAs) was carried out. Binding affinities of the peptides for a hairpin RNA derived from P22 phage were dependent on the types and positions of the NBA units they have. Some NBA peptides bound to the wild-type RNA or its mutant with high affinity and high specificity compared with the native P22 N peptide. These results indicate that the NBA units on the peptides interact with the RNA bases in a specific manner. It is demonstrated that the de novo design of peptides with the NBA units is an effective way to construct novel RNA-binding molecules.

Amino Acids↗
PubMed2003

RNA aptamers targeted to domain II of hepatitis C virus IRES that bind to its apical loop region.

The internal ribosome entry site (IRES) is important for translation of hepatitis C virus (HCV) mRNA and has a unique RNA structure containing conserved domains I to IV. To investigate the function of domain II, we selected RNA aptamers that bind to domain II of HCV IRES by applying a simple and convenient selection method using a hybridized tag for fixing domain II RNA on magnetic beads instead of synthesizing long RNA. In addition, we employed surface plasmon resonance (SPR) technology to measure the binding affinity of each generation and to obtain detailed kinetic constants. The selected aptamers have a consensus sequence, 5'-UAUGGCU-3', which is complementary to the apical loop of domain II. The loop-loop interaction between the consensus sequence and domain II was confirmed by mutagenesis and nuclease mapping analyses. Binding affinities were dependent on the local structure containing the conserved sequence. The aptamers could inhibit IRES-dependent translation.

Base Sequence↗