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At least 1,801 records · Page 100Linked to original sources
PubMed1993

Identification of unusual RNA folding patterns encoded by bacteriophage T4 gene 60.

A 50-nucleotide (nt) untranslated region (coding gap sequence) that interrupts the amino acid coding sequence in T4 gene 60, plus an additional 5 nt upstream and another 3 nt downstream from the gap sequence, shows unusual folding patterns according to RNA structure prediction. A predicted highly stable and significant hairpin structure in the 5' half of the gap sequence and a plausible tertiary structural element computed in the 3' part of the gap sequence seem significant by statistical tests on the wild-type (wt) sequence. This feature is absent in insertion, deletion and substitution variants of the gap sequence, in which template activities are markedly lower than that of the wt. The proposed feature is consistent with currently available data showing that the translational bypass of the coding gap is correlated with a stop codon involved in a stem-loop structure folded in the gap sequence. We suggest that the role of this segment in 'ribosomal bypass' of a portion of the mRNA sequence is a property of its special folded structure.

Bacteriophage T4↗
PubMed2002

Identification and characterization of bacterial class E group II introns.

Group II introns are catalytic RNAs and mobile genetic elements. Phylogenetic characterization of group II intron-encoded reverse transcriptases (RTs) established seven classes: the mitochondrial class, chloroplast-like classes 1 and 2, and bacterial classes A, B, C, and D. In this study, we identified and characterized a new bacterial class of group II introns, bacterial class E, on the basis of phylogenetic analysis of the intron-encoded protein (IEP) RT and determination of a consensus intron RNA structure.

Bacteria↗
PubMed2005

Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints.

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

Amino Acid Motifs↗
PubMed2026

Clinical, genetic and bioinformatic analysis of Saudi families with Joubert syndrome and related disorders.

BACKGROUND: Joubert syndrome and related disorders (JSRD) are clinically and genetically heterogeneous ciliopathies caused by pathogenic variants in over 40 genes, mainly encoding ciliary proteins. However, data on JSRD in the Saudi population remain limited. This study aimed to identify novel and reported JSRD-causing variants in Saudi families and analyze their potential functional impact on protein structure using advanced computational tools. METHODS: Patients with genetically or clinically confirmed/suspected JSRD were recruited according to ethical protocols. Clinical data were collected, and exome sequencing was performed, followed by Sanger validation of identified variant. Pathogenicity was assessed using bioinformatics tools, while conservation, expression and RNA structure analyses were conducted to evaluate the potential functional consequences. RESULTS: Homozygous variants were identified in three consanguineous Saudi families, including a novel stop-gain variant in KIF7 (c.2992 C > T; p.(Gln998Ter)), and novel splice-site variant in CEP104 (c.489 + 1G > A) and a previously reported splice-site variant in TMEM237 (c.869 + 1G > A). All variants were predicted to be pathogenic as per ACMG criteria and located in highly conserved regions, suggesting potential functional impact. RNA analysis suggested possible alterations in folding. CONCLUSION: This study identified three pathogenic variants in JSRD-related genes in Saudi families, including two novel variants. These findings expand the variant spectrum for JSRD, particularly in the Saudi population. Establishing a comprehensive regional variant database is essential for improving molecular diagnosis and genetic counseling in population with high consanguinity. Finally, these results highlight the need for further functional studies to validate their pathogenicity and elucidate their role in JSRD pathogenesis.

Humans↗
PubMed2003

Characterization of the caprine arthritis encephalitis virus (CAEV) rev N-terminal elements required for efficient interaction with the RRE.

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C.

Amino Acid Sequence↗
PubMed2003

HMM sampling and applications to gene finding and alternative splicing.

The standard method of applying hidden Markov models to biological problems is to find a Viterbi (maximal weight) path through the HMM graph. The Viterbi algorithm reduces the problem of finding the most likely hidden state sequence that explains given observations, to a dynamic programming problem for corresponding directed acyclic graphs. For example, in the gene finding application, the HMM is used to find the most likely underlying gene structure given a DNA sequence. In this note we discuss the applications of sampling methods for HMMs. The standard sampling algorithm for HMMs is a variant of the common forward-backward and backtrack algorithms, and has already been applied in the context of Gibbs sampling methods. Nevetheless, the practice of sampling state paths from HMMs does not seem to have been widely adopted, and important applications have been overlooked. We show how sampling can be used for finding alternative splicings for genes, including alternative splicings that are conserved between genes from related organisms. We also show how sampling from the posterior distribution is a natural way to compute probabilities for predicted exons and gene structures being correct under the assumed model. Finally, we describe a new memory efficient sampling algorithm for certain classes of HMMs which provides a practical sampling alternative to the Hirschberg algorithm for optimal alignment. The ideas presented have applications not only to gene finding and HMMs but more generally to stochastic context free grammars and RNA structure prediction.

Algorithms↗
PubMed2004

Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites.

Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.

Base Sequence↗
PubMed1993

Epstein-Barr virus gene expression in oral hairy leukoplakia.

A combination of Northern blotting and sequencing of clones from a cDNA library constructed using RNA isolated from oral hairy leukoplakia (OHL) has been used to study Epstein-Barr virus (EBV) gene expression in this AIDS-associated lesion, the only accessible source of in vivo-replicating EBV. Because of the limited amount of RNA available, Northern blotting was only useful for detection of very abundant EBV transcripts in the OHL biopsies. Analysis of cDNA clones containing the BdRF1, BCRF1, gp350/220, BARF0, and BKRF4 reading frames and further characterization of RNA structures spanning BCRF1 (viral IL-10) in lymphocytes infected with EBV has provided a preliminary comparison of some virus replicative gene expression in its two main host cell types. No expression of EBNA-1, EBNA-2, or EBNA-3A RNA was detected in the oral hairy leukoplakia cDNA library.

Animals↗
PubMed2003

A tumour-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro.

BACKGROUND: The rck/p54 protein of 473 amino acids belongs to the family of DEAD-box/putative RNA helicase proteins. DEAD-box proteins have been implicated in a wide variety of cellular processes ranging from the initiation of protein synthesis and ribosome biosynthesis to premRNA splicing by means of modifying the RNA structure. Our previous data suggested that rck/p54 positively affected the translation initiation of c-myc mRNA. RESULTS: The data obtained from morphological studies and surface plasmon resonance assays clearly indicated that the protein specifically bound to c-myc RNA transcripts (RNAs) and exhibited RNA unwinding activity toward c-myc RNAs in the presence of ATP in vitro. Experiments using a deletion mutant of rck/p54 retaining only its N-terminal 289 amino acids demonstrated that the deleted C-terminal 184 amino acid domain is involved in the RNA unwinding activity. CONCLUSION: These findings strongly suggest that rck/p54 may play an important role in translation initiation by restructuring mRNAs even in the cell and contribute to carcinogenesis.

DEAD-box RNA Helicases↗
PubMed2006

The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain.

Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.

Amino Acid Sequence↗
PubMed1998

Characterization of Toscana virus-defective interfering particles generated in vivo.

Toscana (TOS) virus stocks strongly interfering with standard virus replication were obtained by sequential passages of virus in suckling mouse brain. Characterization of viral RNAs in these stocks showed the presence of a heterogeneous population of defective RNA molecules derived from the L genomic segment, in both nucleocapsid (NC) and messenger RNAs, suggesting that these molecules could be replicated, assembled, and transcribed. Subgenomic RNAs from the L segment but not from the S or M segments were found in cells infected with these stocks. Defective RNA molecules interfered with virus replication and retained 5' and 3' genomic termini. Nucleotide sequence analysis of some cloned defective interfering (DI) RNAs revealed they contained one or more internal deletions reducing their length to 7-13% of the full-length L segment. An identical sequence motif, of variable length, was found at both terminal sites of the RNA junction on standard L sequences. This motif was retained only in one copy in the subgenomic RNA. These results are consistent with the generation of TOS virus DI particles in vivo and suggest that the defective genomic RNAs could be generated by polymerase jumping from a sequence to an identical one spatially closed because of the RNA structure.

Animals↗
PubMed2004

Conserved regulatory motifs in bacteria: riboswitches and beyond.

We present a computational approach that identifies regulatory elements conserved across phylogenetically distant organisms. Intergenic regulatory regions were clustered by orthology of the adjacent genes, and an iterative process was applied to search for significant motifs, enabling new elements of the putative regulon to be added in each cycle. With this approach, we identified highly conserved riboswitches and the Gram positive T-box. Interestingly, we identified many other regulatory systems that appear to depend on conserved RNA structures.

Bacteria↗
PubMed2005

Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation.

Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5' cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.

5' Untranslated Regions↗
PubMed1997

Mg2+ dependence of the structure and thermodynamics of wheat germ and lupin seeds 5S rRNA.

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg2+ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements. Increased concentrations of Mg2+ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na+ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E" hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg2+.

Base Sequence↗
PubMed2006

Unusual mechanical stability of a minimal RNA kissing complex.

By using optical tweezers, we have investigated the mechanical unfolding of a minimal kissing complex with only two G.C base pairs. The loop-loop interaction is exceptionally stable; it is disrupted at forces ranging from 7 to 30 pN, as compared with 14-20 pN for unfolding hairpins of 7 and 11 bp. By monitoring unfolding/folding trajectories of single molecules, we resolved the intermediates, measured their rate constants, and pinpointed the rate-limiting steps. The two hairpins unfold only after breaking the intramolecular kissing interaction, and the kissing interaction forms only after the folding of the hairpins. At forces that favor the unfolding of the hairpins, the entire RNA structure is kinetically stabilized by the kissing interaction, and extra work is required to unfold the metastable hairpins. The strong mechanical stability of even a minimal kissing complex indicates the importance of such loop-loop interactions in initiating and stabilizing RNA dimers in retroviruses.

Base Sequence↗
PubMed2001

Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA.

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.

Amino Acid Sequence↗
PubMed1986

Conformational studies of nucleic acids: IV. The conformational energetics of oligonucleotides: d(ApApApA) and ApApApA.

Utilizing a new method for modeling furanose pseudorotation (D. A. Pearlman and S.-H. Kim, J. Biomol. Struct. Dyn. 3, 85 (1985)) and the empirical multiple correlations between nucleic acid torsion angles we derived in the previous report (D. A. Pearlman and S.-H. Kim, previous paper in this issue), we have made an energetic examination of the entire conformational spaces available to two nucleic acid oligonucleotides: d(ApApApA) and ApApApA. The energies are calculated using a semi-empirical potential function. From the resulting body of data, energy contour map pairs (one for the DNA molecule, one for the RNA structure) have been created for each of the 21 possible torsion angle pairs in a nucleotide repeating unit. Of the 21 pairs, 15 have not been reported previously. The contour plots are different from those made earlier in that for each point in a particular angle-angle plot, the remaining five variable torsion angles are rotated to the values which give a minimum energy at this point. The contour maps are overall quite consistent with the experimental distribution of oligonucleotide data. A number of these maps are of particular interest: delta (C5'-C4'-C3'-O3')-chi (O4'-C1'-N9-C4), where the energetic basis for an approximately linear delta-chi correlation can be seen: zeta (C3'-O3'-P-O5')-delta, in which the experimentally observed linear correlation between zeta and delta in DNA(220 degrees less than zeta less than 280 degrees) is clearly predicted; zeta-epsilon (C4'-C3'-O3'-P), which shows that epsilon increases with decreasing zeta less than 260 degrees; alpha (O3'-P-O5'-C5')-gamma (O5'-C5'-C4'-C3') where a clear linear correlation between these angles is also apparent, consistent with experiment; and several others. For the DNA molecule studied here, the sugar torsion delta is predicted to be the most flexible, while for the RNA molecule, the greatest amount of flexibility is expected to reside in alpha and gamma. Both the DNA and RNA molecules are predicted to be highly polymorphic. Complete energy minimization has been performed on each of the minima found in the energy searches and the results further support this prediction. Possible pathways for B-form to A-form DNA interconversion suggested by the results of this study are discussed. The results of these calculations support use of the new sugar modeling technique and torsion angle correlations in future conformational studies of nucleic acids.

DNA↗
PubMed2005

An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs.

Riboswitches are highly structured RNA elements that control the expression of many bacterial genes by binding directly to small metabolite molecules with high specificity and affinity. In Bacillus subtilis, two classes of riboswitches have been described that discriminate between guanine and adenine despite an extremely high degree of homology both in their primary and secondary structure. We have identified intermolecular base triples between both purine ligands and their respective riboswitch RNAs by NMR spectroscopy. Here, specificity is mediated by the formation of a Watson-Crick base pair between the guanine ligand and a C residue or the adenine ligand and a U residue of the cognate riboswitch RNA, respectively. In addition, a second base-pairing interaction common to both riboswitch purine complexes involves a uridine residue of the RNA and the N3/N9 edge of the purine ligands. This base pairing is mediated by a previously undescribed hydrogen-bonding scheme that contributes to the affinity of the RNA-ligand interaction. The observed intermolecular hydrogen bonds between the purine ligands and the RNA rationalize the previously observed change in specificity upon a C to U mutation in the core of the purine riboswitch RNAs and the differences in the binding affinities for a number of purine analogs.

Adenine↗

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