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At least 847 records · Page 47Linked to original sources
PubMed1994

Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus.

Human immunodeficiency virus type 1 (HIV-1) RNA contains an extended hairpin structure at the 5' end (the TAR element) that is essential for viral replication. The upper part of the stem-loop structure binds the virally encoded transcriptional activator protein Tat and cellular co-factors, but no clear function for the lower stem region has been established. Here, we report that mutant HIV-1 viruses with base substitutions in the lower stem region are dead, most likely at the level of transcription from an integrated provirus. By using large amounts of these mutant DNA constructs for transfections, revertant viruses with a great variety of genetic changes (point mutations, short deletions) could be isolated in prolonged culture experiments that lasted over 6 months. The pattern and evolution of these changes supported the notion that base-pairing of the lower stem region is essential for optimal HIV-1 replication. The functional and genetic plasticities of this RNA domain and the HIV-1 long terminal repeat promoter are discussed.

Base Composition↗
PubMed1994

RNA polymerase: structural determinant of the chromatin loop and the chromosome.

Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a stationary polymerase; individual polymerases tie the chromatin fibre into loops and clusters of polymerases determine the basic structure of the interphase and metaphase chromosome. RNA polymerase is then both a player and a manager of the chromosome loop.

Artifacts↗
PubMed2005

Quantification of the differences between quenched and annealed averaging for RNA secondary structures.

The analytical study of a disordered system is usually difficult due to the necessity to perform a quenched average over the disorder. Thus, one may resort to the easier annealed ensemble as an approximation to the quenched system. In the study of RNA secondary structures, we explicitly quantify the deviation of this approximation from the quenched ensemble by looking at the correlations between neighboring bases. This quantified deviation then allows us to propose a constrained annealed ensemble which predicts some physical quantities much closer to the results of the quenched ensemble without becoming technically intractable.

Binding Sites↗
PubMed1990

Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains.

The five small nuclear RNAs (snRNAs) involved in mammalian pre-mRNA splicing (U1, U2, U4, U5, and U6) are well conserved in length, sequence, and especially secondary structure. These five snRNAs from Saccharomyces cerevisiae show notable size and sequence differences from their metazoan counterparts. This is most striking for the large S. cerevisiae U1 and U2 snRNAs, for which no secondary structure models currently exist. Because of the importance of U1 snRNA in the early steps of "spliceosome" assembly, we wanted to compare the highly conserved secondary structure of metazoan U1 snRNA (approximately 165 nucleotides) with that of S. cerevisiae U1 snRNA (568 nucleotides). To this end, we have cloned and sequenced the U1 gene from two other yeast species possessing large U1 RNAs. Using computer-derived structure predictions, phylogenetic comparisons, and structure probing, we have arrived at a secondary structure model for S. cerevisiae U1 snRNA. The results show that most elements of higher eukaryotic U1 snRNA secondary structure are conserved in S. cerevisiae. The hundreds of "extra" nucleotides of yeast U1 RNA, also highly structured, suggest that large insertions and/or deletions have occurred during the evolution of the U1 gene.

Base Sequence↗
PubMed2001

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex.

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

Base Sequence↗
PubMed1991

Human T-cell leukemia virus type II Rex binding and activity require an intact splice donor site and a specific RNA secondary structure.

The human T-cell leukemia virus type II (HTLV-II) regulatory protein Rex augments cytoplasmic levels of unspliced gag-pol mRNA by acting through a Rex-responsive element (RxRE) in the long terminal repeat. Purified Rex protein binds to long terminal repeat mRNA. Here, using an immunobinding assay to measure the binding of Rex protein to mutated RxRE RNAs, we show that efficient Rex binding requires a stem-bulge-loop RNA secondary structure (nucleotides [nt] 465 to 500) and specific sequences both within the stem-bulge (nt 470 to 476) and within a conserved upstream splice donor site (nt 449 to 455). Rex function in a transient transfection expression system correlates with Rex protein-RxRE RNA binding. The ability of HTLV-II Rex to interact directly with the HTLV-II splice donor site suggests that HTLV-II Rex may increase expression of unspliced gag-pol mRNA, in part, by inhibiting splicing.

Base Sequence↗
PubMed2000

Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA.

Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5'-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.

Animals↗
PubMed2000

Crystal structure of RNA 3'-terminal phosphate cyclase, a ubiquitous enzyme with unusual topology.

BACKGROUND: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea. They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate. These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes. RESULTS: The high-resolution structure of the Escherichia coli RNA 3'-terminal phosphate cyclase was determined using multiwavelength anomalous diffraction. Two orthorhombic crystal forms of E. coli cyclase (space group P2(1)2(1)2(1) and P2(1)2(1)2) were used to solve and refine the structure to 2.1 A resolution (R factor 20.4%; R(free) 27.6%). Each molecule of RNA cyclase consists of two domains. The larger domain contains three repeats of a folding unit comprising two parallel alpha helices and a four-stranded beta sheet; this fold was previously identified in translation initiation factor 3 (IF3). The large domain is similar to one of the two domains of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvyl transferase. The smaller domain uses a similar secondary structure element with different topology, observed in many other proteins such as thioredoxin. CONCLUSIONS: The fold of RNA cyclase consists of known elements connected in a new and unique manner. Although the active site of this enzyme could not be unambiguously assigned, it can be mapped to a region surrounding His309, an adenylate acceptor, in which a number of amino acids are highly conserved in the enzyme from different sources. The structure of E. coli cyclase will be useful for interpretation of structural and mechanistic features of this and other related enzymes.

Amino Acid Sequence↗
PubMed1984

Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region.

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.

DNA-Directed RNA Polymerases↗
PubMed1992

Mutagenesis of the iron-regulatory element further defines a role for RNA secondary structure in the regulation of ferritin and transferrin receptor expression.

Within the 5'-untranslated region of ferritin mRNAs, there is a conserved region of 28 nucleotides (nt) (the iron regulatory element (IRE)) that binds a protein (the IRE-binding protein (IRE-BP)) involved in the iron regulation of ferritin mRNA translation. We have examined the role of RNA secondary structure on the interaction of the IRE with the IRE-BP. First, the rat light ferritin IRE possesses a structure similar to that of the bullfrog heavy ferritin IRE (Wang, Y.-H., Sczekan, S. R., and Theil, E. C. (1990) Nucleic Acids Res. 18, 4463-4468). This includes an extended stem, interrupted at various points by bulge nucleotides and a 6-nt single-stranded loop (CAGUGU) at its top. Computer predictions and mapping results suggest the presence of a 3-nt (UGC) bulge 5 bases 5' of the loop in the rat IRE. Second, disruption of the base pairing in the upper stem alters IRE secondary structure and reduces the affinity with which the IRE-BP binds the IRE. Third, increasing the size of the loop or the distance between the UGC bulge and the loop reduces the IRE/IRE-BP interaction. Our results indicate that several aspects of IRE secondary structure are important for its high affinity binding to the IRE-BP.

Animals↗
PubMed2006

Characterization of the RNA chaperone activity of hantavirus nucleocapsid protein.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein, encoded by the smallest of the three genome segments (S), has nonspecific RNA chaperone activity. This activity results in transient dissociation of misfolded RNA structures, may be required for facilitating correct higher-order RNA structure, and may function in viral genome replication. We carried out a series of experiments to further characterize the ability of N to dissociate RNA duplexes. As might be expected, N dissociated RNA duplexes but not DNA duplexes or RNA-DNA heteroduplexes. The RNA-destabilizing activity of N is ATP independent, has a pH optimum of 7.5, and has an Mg(2+) concentration optimum of 1 to 2 mM. N protein is unable to unwind the RNA duplexes that are completely double stranded. However, in the presence of an adjoining single-stranded region, helix unwinding takes place in the 3'-to-5' direction through an unknown mechanism. The N protein trimer specifically recognizes and unwinds the terminal panhandle structure in the viral RNA and remains associated with unwound 5' terminus. We suggest that hantaviral nucleocapsid protein has an active role in hantaviral replication by working cooperatively with viral RNA polymerase. After specific recognition of the panhandle structure by N protein, the unwound 5' terminus likely remains transiently bound to N protein, creating an opportunity for the viral polymerase to initiate transcription at the accessible 3' terminus.

Adenosine Triphosphate↗
PubMed1986

DNA-RNA hybrid secondary structures.

DNA-RNA and DNA-DNA duplexes are even more polymorphic than observed previously. DNA-RNA hybrids can have secondary structures like A-DNA or A-RNA, but double helices of the synthetic DNA-RNA hybrids poly(dA) X poly(rU) and poly(dI) X poly(rC), respectively, form 11-fold and 10-fold double-helical structures in which the two chains have quite different conformations. Extensive X-ray fiber diffraction analyses show that in both structures the DNA chains have C-2'-endo-puckered furanose rings, while the anti-parallel RNA chains have C-3'-endo-puckered rings. The bidirectional properties of such duplexes may be important in the transfer of biological information from nucleic acids.

Base Composition↗
PubMed1997

RNA secondary structure as a reusable interface to biological information resources.

The dissemination of biological information has become critically dependent on the Internet and World Wide Web (WWW), which enable distributed access to information in a platform independent manner. The mode of interaction between biologists and on-line information resources, however, has been mostly limited to simple interface technologies such has hypertext links, tables and forms. The introduction of platform-independent runtime environments facilitates the development of more sophisticated WWW-based user interfaces. Until recently, most such interfaces have been tightly coupled to the underlying computation engines, and not separated as reusable components. We believe that many subdisciplines of biology have intuitive and familiar graphical representations of knowledge that can serve as multipurpose user interface elements. We call such graphical idioms "domain graphics". In order to illustrate the power of such graphics, we have built a reusable interface based on the standard two dimensional (2D) layout of RNA secondary structure. The interface can be used to represent any pre-computed layout of RNA, and takes as a parameters the sets of actions to be performed as a user interacts with the interface. It can provide to any associated application program information about the base, helix, or subsequence selected by the user. We show the versatility of this interface by using it as a special purpose interface to BLAST, Medline and the RNA MFOLD search/compute engines. These demonstrations are available at: http://www-smi.stanford.edu/projects/helix/pubs/ gene-combis-96/

Base Sequence↗
PubMed2005

Consensus shapes: an alternative to the Sankoff algorithm for RNA consensus structure prediction.

MOTIVATION: The well-known Sankoff algorithm for simultaneous RNA sequence alignment and folding is currently considered an ideal, but computationally over-expensive method. Available tools implement this algorithm under various pragmatic restrictions. They are still expensive to use, and it is difficult to judge if the moderate quality of results is because of the underlying model or to its imperfect implementation. RESULTS: We propose to redefine the consensus structure prediction problem in a way that does not imply a multiple sequence alignment step. For a family of RNA sequences, our method explicitly and independently enumerates the near-optimal abstract shape space, and predicts as the consensus an abstract shape common to all sequences. For each sequence, it delivers the thermodynamically best structure which has this common shape. Since the shape space is much smaller than the structure space, and identification of common shapes can be done in linear time (in the number of shapes considered), the method is essentially linear in the number of sequences. Our evaluation shows that the new method compares favorably with available alternatives. AVAILABILITY: The new method has been implemented in the program RNAcast and is available on the Bielefeld Bioinformatics Server. CONTACT: jreeder@TechFak.Uni-Bielefeld.DE, robert@TechFak.Uni-Bielefeld.DE SUPPLEMENTARY INFORMATION: Available at http://bibiserv.techfak.uni-bielefeld.de/rnacast/supplementary.html

Algorithms↗
PubMed2006

The prediction of the wild-type telomerase RNA pseudoknot structure and the pivotal role of the bulge in its formation.

In this study, the three-dimensional structure of the wild-type human telomerase RNA pseudoknot was predicted via molecular modeling. The wild-type pseudoknot structure is then compared to the recent NMR solution structure of the telomerase pseudoknot, which does not contain the U177 bulge. The removal of the bulge from the pseudoknot structure results in higher stability and significant reduction of activity of telomerase. We show that the effect of the bulge on the structure results in a significant transformation of the pseudoknot junction region where the starting base pairs are disrupted and unique triple base pairs are formed. We found that the formation of the junction region is greatly influenced by interactions of the U177 bulge with loop residues and rotation of residue A174. Moreover, this is the first study to our knowledge where a structure as complex as the pseudoknot has been solved by purely theoretical methods.

Base Pairing↗
PubMed2000

RNA secondary structures of the bacteriophage phi6 packaging regions.

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Bacteriophage phi 6↗
PubMed1986

Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector.

Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus-strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle.

Adenoviruses, Human↗
PubMed1994

Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI.

The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.

Adenoviruses, Human↗