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At least 451 records · Page 25Linked to original sources
PubMed1992

Biochemical and NMR spectroscopy evidence for a new tertiary A-U base pair in lupin ribosomal 5 S RNA structure.

The new model for the tertiary structure of ribosomal 5 S rRNA from plants recently proposed by some of us has been already supported by RNase H digestions in the presence of complementary oligodeoxynucleotides. These results are confirmed now by the new biochemical and NMR spectroscopy data. Diethylpyrocarbonate (DEP) and monoperphthalic acid (MPA) are the reagents with the high specificity toward single-stranded adenosine residues. Our experiments clearly show that under native conditions adenosine 100 (A100) of lupin 5 S rRNA is not available for reaction toward these reagents. However under denaturing conditions this residue reacts with DEP and MPA. The detailed analysis of the lupin 5 S rRNA by NMR spectra provide the data on the specific interaction of A100-U53. Thus, we have seen for the first time the NMR signal due to the A100-U53 tertiary base pair, which as we believe, stabilizes interactions between loops B and E.

Adenine↗
PubMed1998

Translational attenuation of the Bacillus subtilis spo0B cistron by an RNA structure encompassing the initiation region.

The spo0B gene, which exists as an operon with the obg gene, is required to initiate sporulation (stage 0) of Bacillus subtilis . This gene encodes a phosphotransferase in the multicomponent phosphorelay system. We here report the novel finding that a spo0B 5'-terminal SLR (stem-loop structure sequestering ribosome binding sequence; ACUCCUAA-X16-UUG GGAG U, Delta G = -8.71 kcal/mol) attenuated spo0B translation. The spo0B gene was efficiently transcribed but Spo0B protein was not overproduced in Escherichia coli when spo0B was induced using expression vectors carrying the SLR- spo0B region under control of the tac promoter. Deletion of the SLR from the vectors resulted in overexpression of spo0B . Therefore, to characterize expression of spo0B with a SLR in B.subtilis we constructed transcriptional and translational lacZ fusions combined with the spo0B 5'-terminal region with a deleted or mutagenized SLR. These constructs were subsequently introduced into B.subtilis as multiple and single copies, then beta-galactosidase activities were measured. The possible SLR also functioned as a negative cis element in B.subtilis. Furthermore, B.subtilis strain 1S16 (spo0B136) lysogenized straight phiCD0B-S and -W, harboring spo0B with mutagenized SLRs that were more (Delta G = -14.0 kcal/mol) and less-stable (Delta G = -1.31 kcal/mol) compared with the wild-type, exhibited null and wild-type sporulation respectively. These results indicate that the spo0B 5'-SLR affects spo0B gene expression for sporulation but that low expression of spo0B through the wild-type SLR was sufficient to initiate sporulation in B.subtilis.

Amino Acid Sequence↗
PubMed1991

Evolution of messenger RNA structure and regulation in the genus Mus: the androgen-inducible RP2 mRNAs.

The RP2 gene is one of several genes that are regulated by androgens in the mouse kidney. Previous studies have demonstrated that androgen inducibility of RP2 transcription varies among species within the genus Mus, indicating extensive evolutionary modification of the participating regulatory elements. Thus, while a five-fold induction of transcription occurs in M. domesticus, none is detectable in M. hortulanus or M. caroli. In the present paper, we have sequenced cDNAs representing the RP2 mRNAs of M. caroli and M. saxicola and have compared them with each other and with M. domesticus. Several findings from the sequence comparisons indicate that the encoded 41-kD polypeptide is physiologically functional. First, divergence within noncoding regions of the mRNAs exceeds that within coding regions. Second, the 357-codon open reading frame has been maintained among the species, with approximately 90% of the amino acid replacements being conservative. Finally, substitution rates at synonymous sites within the coding regions are from twofold to threefold greater than those at nonsynonymous sites. The genetic elements responsible for variations in RP2 inducibility among species were studied by cis/trans analyses of mice heterozygous for RP2 alleles, using a primer extension assay to measure expression of species-specific mRNAs. The results show that the absence of transcriptional induction in M. hortulanus is due to a cis-acting genetic element, while that in M. caroli is due to a trans-acting element. Thus, the androgen-resistant RP2 phenotypes of these two species derive from distinct genetic events.

Animals↗
PubMed1992

Identifying constraints on the higher-order structure of RNA: continued development and application of comparative sequence analysis methods.

Comparative sequence analysis addresses the problem of RNA folding and RNA structural diversity, and is responsible for determining the folding of many RNA molecules, including 5S, 16S, and 23S rRNAs, tRNA, RNAse P RNA, and Group I and II introns. Initially this method was utilized to fold these sequences into their secondary structures. More recently, this method has revealed numerous tertiary correlations, elucidating novel RNA structural motifs, several of which have been experimentally tested and verified, substantiating the general application of this approach. As successful as the comparative methods have been in elucidating higher-order structure, it is clear that additional structure constraints remain to be found. Deciphering such constraints requires more sensitive and rigorous protocols, in addition to RNA sequence datasets that contain additional phylogenetic diversity and an overall increase in the number of sequences. Various RNA databases, including the tRNA and rRNA sequence datasets, continue to grow in number as well as diversity. Described herein is the development of more rigorous comparative analysis protocols. Our initial development and applications on different RNA datasets have been very encouraging. Such analyses on tRNA, 16S and 23S rRNA are substantiating previously proposed associations and are now beginning to reveal additional constraints on these molecules. A subset of these involve several positions that correlate simultaneously with one another, implying units larger than a basepair can be under a phylogenetic constraint.

Base Sequence↗
PubMed2004

RNA 3D structure prediction: (1) assessing rna 3D structure similarity from 2D structure similarity.

Computational techniques for 3D structure prediction of proteins, the holy grail of bioinformatics, have undergone major developments in recent years, geared by international cooperation and competition with CASP (Critical Assessment of Structure Prediction Techniques) like contests to improve and refine them. Although straightforward extrapolation of these methodologies for the prediction of the 3D structures of other similarly relevant bio macromolecules may not be too compelling due mostly to the intrinsic differences in constitution, nature, and function between them, the conceptual framework underlying most of those techniques applied to the development of similar computational techniques in structural biology can lead to efficient systems for prediction of the 3D structure of other bio-macromolecules. One of them is the development of rational methodologies to model RNA 3D structures from the sequence of nucleotides composing them. In this paper we establish the fundamentals of a methodology to thread a sequence of nucleotides into a set of 3D fragments extracted from a data base expressly developed for this purpose. The technique is based on a newly implemented algorithm for extraction of 3D fragments by comparison of secondary structures of RNA. The result is a highly efficient system to produce a set of fragments from which entire RNA structure for the given nucleotide sequence can be built.

Algorithms↗
PubMed2005

The effect of RNA secondary structures on RNA-ligand binding and the modifier RNA mechanism: a quantitative model.

RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small "modifier RNAs" to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements. We discuss our model and recent experimental findings demonstrating the effectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.

Algorithms↗
PubMed1997

RNA seeing double: close-packing of helices in RNA tertiary structure.

Structured RNA molecules play essential roles in RNA processing, chromosome maintenance and protein biosynthesis. RNA necessarily uses different strategies than proteins for folding and assembly of complex architectures. The RNA-folding problem is largely an issue of helical packing: how does RNA organize and pack short, double-helical segments to produce active sites and recognition motifs for proteins? Noncanonical base pairs, metal ions and 2'-hydroxyl groups are key elements in RNA higher-order structure formation.

Base Composition↗
PubMed1993

Modeling the three-dimensional structure of RNA.

The limited number of RNA structures determined by X-ray crystallography and NMR spectroscopy compels the use of experimental and theoretical methods that are less precise to obtain information on RNA conformation. RNA flexibility, a consequence of rotational freedom about seven intra- and internucleotide bonds, is unfortunately of such magnitude that these alternate techniques fall short of providing sufficient information to build robust tertiary structures. Various RNA modeling methods, described herein, permit the organization of this structural data to the form of three-dimensional structures. Interactive computer graphics techniques, for example, have generated several useful models. Also, conventional computer algorithms involving the minimization of empirical energy functions, previously limited to small molecules, are giving way to methods able to handle much larger molecules. Modified distance geometry and molecular mechanics algorithms, using simplified "pseudoatom" representations, can generate structures consistent with input data. A constraint satisfaction algorithm combined with discrete representations of nucleotide conformations systematically explores poorly defined regions of a molecule yielding all-atom representations, but requires enough structural constraints to avoid a computational explosion.

Algorithms↗
PubMed1991

Modeling RNA secondary structures. I. Mathematical structural model for predicting RNA secondary structures.

A mathematical model for analyzing the secondary structures of RNA is developed that is based on the connection matrix associated with the planar p-h graph. The classification of the elementary structures allows the introduction of the basis of structural space from which to build the global secondary structure. All admissible solutions belong to the configuration space and can be obtained directly from its basis.

Algorithms↗
PubMed1990

Synthesis of RNA having cap structure.

RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O-methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA.

Amino Acid Sequence↗
PubMed1984

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review.

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.

Base Sequence↗
PubMed2006

Displaying 3D data on RNA secondary structures: coloRNA.

RNA performs a variety of diverse functions and therefore must adopt many different three-dimensional conformations. The number and complexity of RNA structures that are currently available are steadily increasing, necessitating the generation of versatile structure visualization tools. Here, we describe a new RNA secondary and tertiary structure visualization tool, the display program coloRNA. This program colors each nucleotide in a secondary structure schematic according to the value of an assigned property of the corresponding backbone phosphate group, such as the distance between corresponding residues in two atomic models of the same RNA molecule. To assist in analyzing tertiary structure, coloRNA also colors nucleotides based on the three-dimensional distances between a user-selected nucleotide and all others. Minimum and maximum thresholds can be used to focus in on, or eliminate, a particular value range. coloRNA can display a user-specified group of nucleotides by outlining the structure in an automatically assigned, but user-changeable color. As an example, we have used coloRNA to analyze a pair of recently published structures of the Escherichia coli 70S ribosome. When coloRNA is used to display the conformational difference between the two structures, the large movement of the small subunit head stands visually out from the background changes in the remaining domains of the small subunit.

Escherichia coli↗
PubMed1998

Global flexibility of tertiary structure in RNA: yeast tRNAPhe as a model system.

The study of RNA structure using x-ray crystallography or NMR has yielded a wealth of detailed structural information; however, such approaches do not generally yield quantitative information regarding long-range flexibility in solution. To address this issue, we describe a solution-based method that is capable of characterizing the global flexibilities of nonhelix elements in RNA, provided that such elements are flanked by helix (e.g., bulges, internal loops, or branches). The "phased tau ratio" method is based on the principle that, for RNA molecules possessing two variably phased bends, the relative birefringence decay times depend on the flexibility of each bend, not simply the mean bend angles. The method is used to examine the overall flexibility of the yeast tRNAPhe core (as unmodified transcript). In the presence of magnesium ions, the tRNA core is not significantly more flexible than an equivalent length of RNA helix. In the absence of divalent ions, the tRNA core gains flexibility under conditions where its secondary structure is likely to be largely preserved. The phased tau ratio approach should be broadly applicable to nonhelix elements in both RNA and DNA and to protein-nucleic acid interactions.

Base Sequence↗