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At least 865 records · Page 48Linked to original sources
PubMed1983

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Splicing of the ribosomal RNA precursor of Tetrahymena is an autocatalytic reaction, requiring no enzyme or other protein in vitro. The structure of the intervening sequence (IVS) appears to direct the cleavage/ligation reactions involved in pre-rRNA splicing and IVS cyclization. We have probed this structure by treating the linear excised IVS RNA under nondenaturing conditions with various single- and double-strand-specific nucleases and then mapping the cleavage sites by using sequencing gel electrophoresis. A computer program was then used to predict the lowest-free-energy secondary structure consistent with the nuclease cleavage data. The resulting structure is appealing in that the ends of the IVS are in proximity; thus, the IVS can help align the adjacent coding regions (exons) for ligation, and IVS cyclization can occur. The Tetrahymena IVS has several sequences in common with those of fungal mitochondrial mRNA and rRNA IVSs, sequences that by genetic analysis are known to be important cis-acting elements for splicing of the mitochondrial RNAs. In the predicted structure of the Tetrahymena IVS, these sequences interact in a pairwise manner similar to that postulated for the mitochondrial IVSs. These findings suggest a common origin of some nuclear and mitochondrial introns and common elements in the mechanism of their splicing.

Base Sequence↗
PubMed1983

Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members.

Two families of fungal mitochondrial introns that include all known sequences have been recognized. These families are now extended to incorporate a plant mitochondrial intron and several introns in chloroplast- and nuclear-encoded rRNA and tRNA precursors. Members of the same family share distinctive sequence stretches and a number of potential RNA secondary structures that would bring these stretches and the intron-exon junctions into relatively close proximity. Using several of these introns which have been extensively studied by either biochemical or genetic means, an attempt is made to integrate the available data into a common picture.

Base Sequence↗
PubMed1989

RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes.

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.

Base Sequence↗
PubMed2005

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions.

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.

Animals↗
PubMed2005

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA.

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.

Animals↗
PubMed1992

5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.

A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR.

Animals↗
PubMed2005

The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints.

Identification of microbial community members in complex environmental samples is time consuming and repetitive. Here, ribosomal sequences and hidden Markov models are used in a novel approach to rapidly assign fungi to their presumptive genera. The ITS1 and ITS2 fragments from a range of axenic, anaerobic gut fungal cultures, including several type strains, were isolated and the RNA secondary structures predicted for these sequences were used to generate a fingerprinting program. The methodology was then tested and the algorithms improved using a collection of environmentally derived sequences, providing a rapid indicator of the fungal diversity and numbers of novel sequence groups within the environmental sample from which they were derived. While the methodology was developed to assist in investigations involving the rumen ecosystem, it has potential generic application in studying diversity and population dynamics in other microbial ecosystems.

Animals↗
PubMed2005

The flavivirus-conserved penta-nucleotide in the 3' stem-loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation.

A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3' untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3' UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5'-CACAG-3' located at the top of the 3' stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication.

Conserved Sequence↗
PubMed1991

The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro.

Although the lengthy 5' nontranslated regions (5'NTRs) of other picornaviral RNAs form highly ordered structures with important functions in viral translation, little is known about the 5'NTR of hepatitis A virus (HAV). We determined the nearly complete 5'NTR nucleotide sequences of two genetically divergent HAV strains (PA21 and CF53) and included these data in a comparative phylogenetic analysis of the HAV 5'NTR. We identified covariant nucleotide substitutions predictive of conserved secondary structures and used this information to develop a model of the 5'NTR secondary structure, which was further refined by thermodynamic predictions and nuclease digestion experiments. According to this model, the 5'NTR comprises six major structural domains. Domains I and II (bases 1 to 95) contain a 5'-terminal hairpin and two stem-loops followed by a single-stranded and highly variable pyrimidine-rich tract (bases 96 to 154). The remainder of the 5'NTR (domains III to VI, bases 155 to 734) contains several complex stem-loops, one of which may form a pseudoknot, and terminates in a highly conserved region containing an oligopyrimidine tract preceding the putative start codon by 13 bases. To determine which structural elements might function as an internal ribosome entry site, RNA transcripts representing the HAV 5'NTR with progressive 5' deletions were translated in rabbit reticulocyte lysates. The translation product was truncated, unprocessed P1 polyprotein. Removal of the 5'-terminal 354 bases of the 5'NTR had little effect on translation. However, deletion to base 447 slightly decreased translation, while deletion to base 533 almost completely abolished it. These data indicate that sequences 3' of base 355 play an important role in the translation mechanism utilized by genomic-length HAV RNA. Significantly, this region shares several conserved structural features with the internal ribosome entry site element of murine encephalomyocarditis virus.

Animals↗
PubMed1985

Purification and subunit structure of RNA polymerases I and II from Dictyostelium discoideum vegetative cells.

A purification procedure to obtain RNA polymerases I (or A) and II (or B) from Dictyostelium discoideum amoeba has been developed. The enzymes were solubilized from purified nuclei and separated by DEAE-Sephadex chromatography. RNA polymerases I and II were further purified by a second chromatography on DEAE-Sephadex followed by chromatographies on phosphocellulose and heparin-sepharose. The specific activities of purified RNA polymerases I and II are 92 units/mg protein and 70 units/mg protein, respectively. The subunit structure of both RNA polymerases were analyzed by polyacrylamide gel electrophoresis under denaturing conditions after glycerol gradient centrifugation of the enzymes. The putative subunits of RNA polymerase I have molecular weights of 180 000, 125 000, 43 000, 40,000, 34 000, 31 000, 25 000, 19 000, 17 000 and 14 000. The putative subunits of RNA polymerase II have molecular weights of 200 000 (170 000), 130 000, 33 000, 25 000, 19 000, 17 000, 15 000, 13 000. There are three polypeptides with common molecular weight in Dictyostelium RNA polymerases I and II. The subunit of 25 000 daltons of both enzymes has common immunological determinants with RNA polymerase II from crustacean Artemia.

Chromatography, Affinity↗
PubMed2001

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA.

Posttranscriptional editing of trypanosome mitochondrial messenger RNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examined. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examined using solution-probing techniques. The mRNA/gRNA crosslinked molecules are efficient substrates for gRNA-directed cleavage. In addition, when the cleavage assay is performed in the presence or absence of additional UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the molecules in a biologically relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide additional support for the predicted secondary structure of interacting gRNA/mRNA pairs.

Animals↗
PubMed1997

High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA.

The structure of the C-terminal RNA recognition domain of ribosomal protein L11 has been solved by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. Although the structure can be considered high resolution in the core, 15 residues between helix alpha 1 and strand beta 1 form an extended, unstructured loop. 15N transverse relaxation measurements suggest that the loop is moving on a picosecond-to-nanosecond time scale in the free protein but not in the protein bound to RNA. Chemical shifts differences between the free protein and the bound protein suggest that the loop as well as the C-terminal end of helix alpha 3 are involved in RNA binding.

Amino Acid Sequence↗
PubMed2004

Structure and RNA interactions of the N-terminal RRM domains of PTB.

The polypyrimidine tract binding protein (PTB) is an important regulator of alternative splicing that also affects mRNA localization, stabilization, polyadenylation, and translation. NMR structural analysis of the N-terminal half of PTB (residues 55-301) shows a canonical structure for RRM1 but reveals novel extensions to the beta strands and C terminus of RRM2 that significantly modify the beta sheet RNA binding surface. Although PTB contains four RNA recognition motifs (RRMs), it is widely held that only RRMs 3 and 4 are involved in RNA binding and that RRM2 mediates homodimerization. However, we show here not only that the RRMs 1 and 2 contribute substantially to RNA binding but also that full-length PTB is monomeric, with an elongated structure determined by X-ray solution scattering that is consistent with a linear arrangement of the constituent RRMs. These new insights into the structure and RNA binding properties of PTB suggest revised models of its mechanism of action.

Amino Acid Sequence↗
PubMed2006

In silico predicted robustness of viroid RNA secondary structures. II. Interaction between mutation pairs.

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as highly branched structures. In a previous study, we used RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. Here we used the same approach to explore the sign and strength of epistasis among pairs of random mutations. We found that antagonistic interactions were more abundant than synergistic ones. However, despite their lower frequency, synergistic interactions tended to be more intense. Mutational robustness and the intensity of epistasis were correlated such that viroid species with large average mutational effects showed stronger antagonistic epistasis, whereas viroids with mild average mutational effects showed weaker antagonistic interactions. The strength of antagonistic epistasis decreased with genome complexity as a consequence of the gained robustness of duplicated genomes. In good agreement with our previous finding of an evolutionary trend toward increased robustness, we now found a trend toward reduced antagonistic epistasis.

Base Pairing↗
PubMed1991

Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure.

Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield. However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter.

Base Sequence↗
PubMed1990

Monomethylated cap structures facilitate RNA export from the nucleus.

RNA export from the nucleus has been analyzed in Xenopus oocytes. U1 snRNAs made by RNA polymerase II were exported into the cytoplasm, while U1 snRNAs synthesized by RNA polymerase III, and therefore with a different cap structure, remained in the nucleus. Export of the polymerase II-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs carrying monomethylguanosine cap structures were rapidly exported, while hypermethylated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable splicing complexes was also significantly delayed by incorporation of a hypermethylated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus.

Animals↗
PubMed1991

Transcriptional elongation by purified RNA polymerase II is blocked at the trans-activation-responsive region of human immunodeficiency virus type 1 in vitro.

It has previously been shown that the human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive region (TAR) is contained in a stem-loop RNA structure. Moreover, the interaction of the RNA secondary structure with Tat, the trans-activator protein, seems to play a role in activation of transcription initiation and in preventing transcription attenuation. In this work, we have studied the ability of the HIV-1 TAR stem-loop to act as a specific attenuation signal for highly purified RNA polymerase II. We developed an in vitro system using dC-tailed DNA fragments of HIV-1 to study transcriptional control in the HIV-1 LTR. We have found that transcription in this system yields an attenuator RNA whose 3' end maps to the end of the TAR stem-loop, approximately 60 to 65 nucleotides downstream of the in vivo initiation site. Furthermore, transcription attenuation occurs only under conditions which cause displacement of the nascent transcript from the template DNA strand, thus allowing the RNA to fold into secondary structure. Evidence is provided that the purified polymerase II indeed recognizes stable RNA secondary structure as an intrinsic attenuation signal. The existence of this signal in the TAR stem-loop suggests that in vivo an antiattenuation factor, probably Tat, alone or in combination with other factors, acts to relieve the elongation block at the HIV-1 attenuation site.

Base Sequence↗
PubMed2003

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs.

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.

3' Untranslated Regions↗