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At least 631 records · Page 35Linked to original sources
PubMed2006

Sub-plastidial localization of two different phage-type RNA polymerases in spinach chloroplasts.

Plant plastids contain a circular genome of approximately 150 kb organized into approximately 35 transcription units. The plastid genome is organized into nucleoids and attached to plastid membranes. This relatively small genome is transcribed by at least two different RNA polymerases, one being of the prokaryotic type and plastid-encoded (PEP), the other one being of the phage-type and nucleus-encoded (NEP). The presumed localization of a second phage-type RNA polymerase in plastids is still questionable. There is strong evidence for a sequential action of NEP and PEP enzymes during plant development attributing a prevailing role of NEP during early plant and plastid development, although NEP is present in mature chloroplasts. In the present paper, we have analysed two different NEP enzymes from spinach with respect to subcellular and intra-plastidial localization in mature chloroplasts with the help of specific antibodies. Results show the presence of the two different NEP enzymes in mature chloroplasts. Both enzymes are entirely membrane bound but, unlike previously thought, this membrane binding is not mediated via DNA. This finding indicates that NEP enzymes are not found as elongating transcription complexes on the template DNA in mature chloroplasts and raises the question of their function in mature chloroplasts.

Amino Acid Sequence↗
PubMed1993

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.

Base Sequence↗
PubMed1994

Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region.

We previously showed that the in vitro transcription of a negatively supercoiled plasmid containing the murine IgA switch region caused the formation of fewer supercoiled conformers of the plasmid due to the presence of a stable RNA.DNA hybrid. Here, we demonstrate that the RNA.DNA hybrid is approximately 140 nucleotides, and it forms regardless of the initial topological state of the transcription template. Transcription of the switch region in a relaxed closed circular plasmid generates positively supercoiled plasmid conformers that revert to their original relaxed state when treated with RNase H. Conformers that have incorporated a stable RNA transcript are also observed when nicked circular and linear plasmids containing the IgA switch sequences are transcribed with 32P-labeled nucleotide triphosphates. Once formed, the RNA.DNA hybrid is stable to both thermal and superhelical stress, tolerating temperatures in excess of 95 degrees C and restraining approximately 12 positive supercoils in the plasmid.

Animals↗
PubMed1984

Transcription of episomal papillomavirus DNA in human condylomata acuminata and Buschke-Löwenstein tumours.

Condylomata acuminata and Buschke-Löwenstein tumours were analysed for the presence of human papillomavirus (HPV) transcripts. HPV DNA and RNA sequences were present in all 13 samples investigated. Ten contained HPV6 and three harboured HPV11. The HPV genomes were found exclusively as extrachromosomal circular molecules. In six biopsy specimens, viral RNA transcripts were not detectable by Northern blot analysis but could be demonstrated in dot blots. From seven HPV6-containing samples it was possible to obtain sufficient amounts of undegraded mRNA. We have found consistently one major species (1.4 kb). Less prominent species of 1.7, 1.85, 2.7 and 3.2 kb, respectively, were also detected. The 3' ends of the HPV6 mRNAs were located between nucleotides 3917 and 4441 in the putative early region and between nucleotides 7232 and 7696 in the putative late region. The arrangement of the 3' termini and the adjacent coding areas within the HPV6 genome show that the RNA species are transcribed from one DNA strand.

Base Sequence↗
PubMed1994

A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

Base Sequence↗
PubMed1984

Hepatitis B virus particles of plasma and liver contain viral DNA-RNA hybrid molecules.

Hepatitis B virions in plasma (Dane particles) are known to contain small circular DNA molecules. The experiments described here indicate that virions in plasma, as well as particles from hepatitis B virus-infected human liver, also contain viral DNA-RNA hybrid molecules, and deoxynucleotides can be incorporated into the DNA of these hybrids by DNA polymerase activities in the virions. Thus, two viral DNA synthetic reactions appear to take place in virions: repair of the single-stranded region of circular DNA molecules and synthesis or elongation of the DNA strand of DNA-RNA hybrid molecules. Centrifugation of virion nucleic acid to equilibrium in Cs2SO4 density gradients revealed the presence of viral DNA-RNA hybrid molecules over a density range of 1.45 to 1.60 g/cm3. Distinct species of hybrid molecules were found with an average density of 1.57 g/cm3 in Dane particles and 1.52 and 1.57 g/cm3 in particles from liver. Fractionation of nucleic acid from Cs2SO4 density gradients by gel electrophoresis demonstrated that the majority of hybrid molecules migrated faster than molecules with the density of pure DNA (1.42 g/cm3). One notable exception was the finding of DNA-RNA hybrid molecules migrating slower than open circular viral DNA. Characterization of viral DNA-RNA hybrids by heat denaturation Cs2SO4 density gradient fractionation, and recombinant M13-HBV single-stranded probe hybridization revealed that the hybrid molecules consisted of viral plus-strand RNA hydrogen bonded to viral minus-strand DNA sequences. Data obtained by pancreatic ribonuclease digestion revealed that the hybrid molecules at density 1.45 to 1.52 g/cm3 contained HBV RNA strands base paired over only part of their length in contrast to the hybrid species at density 1.57 g/cm3 which contained RNA strands apparently base paired over most of their length. Further characterization showed that the hybrid at 1.57 g/cm3 contained genome-length minus-strand viral DNA. The experiments rule out the possibility that the hybrid molecules are transcriptional complexes. Data presented in a companion manuscript indicate that the hybrid molecules may represent intermediates in the synthesis of viral DNA in the endogenous DNA polymerase reaction.

DNA, Viral↗
PubMed1998

Kinetics at a multifunctional RNA active site.

Kinetics of a self-capping RNA, Iso6, have been investigated to constrain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking group or on the RNA. While the number of phosphates in the nucleophile affects capping kinetics, only KM but not kcat is altered. The KM values for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 microM, respectively. A reaction product, pyrophosphate, is also found to strongly inhibit RNA activities through a competitive exchange mechanism with an apparent Ki of 200 nM. Uniquely strong binding of pyrophosphate supports the idea that capping originated by utilization of the initial pyrophosphate leaving group site for capping nucleophiles. In contrast to the nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and kcat by 40%, with little effect on KM. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanisms that explain this apparent rate-limiting chemical step, either dissociation of pyrophosphate to form a metaphosphate monoester intermediate or formation of a circular phosphoramidate intermediate, using an internal RNA nitrogenous group. A single essential Ca ion is required for all activities.

Calcium↗
PubMed2004

Animal-free production of ccc-supercoiled plasmids for research and clinical applications.

The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.

DNA, Superhelical↗
PubMed2001

The DNA form of a retroviroid-like element characterized in cultivated carnation species.

Carnation small viroid-like RNA (CarSV RNA) is a small (275 nt), circular molecule which is unique among plant viroid-like RNAs in having a tandemly repeated homologous DNA. This DNA form was found fused to DNA sequences of carnation etched ring caulimovirus (CERV) in certain Spanish carnation plants. The observation of a growth abnormality consisting of extensive shoot proliferation in cultivated carnations in Hungary prompted the molecular analysis of these plants, in which both CarSV RNA and DNA forms were detected. Several CarSV DNA sequences were characterized in various Dianthus caryophyllus cultivars which were symptomless or showed different symptoms. CarSV DNA forms showing minor sequence heterogeneities and deletions occurred in the same plant. Unit-length CarSV DNA sequences were proven to accumulate in the plant cell nucleus. The plants studied here were not infected by any of the viruses (including CERV) or other cellular pathogens described previously in carnation.

Base Sequence↗
PubMed2026

Circular RNAs orchestrate integrated post-transcriptional responses to combined heat and drought stress in rice.

Circular RNAs (circRNAs) are emerging post-transcriptional regulators, yet their landscape and functional roles in rice under combined abiotic stress remain largely unexplored. Here, we systematically reanalyzed strand-specific RNA-seq data to characterize circRNAs responsive to simultaneous heat and drought stress. Following quality control, read mapping, and dual-algorithm prediction using CIRI2 and CIRCexplorer2, we identified 208 high-confidence circRNAs distributed across all 12 chromosomes. Comparative profiling revealed 83 circRNAs uniquely expressed in control samples, 51 in stressed samples, and 74 shared between conditions, indicating stress-dependent circularization. Junction-read analysis highlighted a spectrum of circularization strength, ranging from highly abundant circRNAs with dominant junction reads to low-confidence candidates masked by linear transcript background. Genomic annotation showed that circRNAs primarily originated from exonic and intergenic regions, with a pronounced negative-strand bias; several genes generated multiple circRNA isoforms via alternative back-splicing. Functional enrichment of host genes suggested involvement in protein folding, nutrient reservoir activity, RNA degradation, and branched-chain amino acid catabolism, implicating roles in stress adaptation and metabolic regulation. Differential expression analysis identified seven circRNAs specifically induced under combined stress conditions. Network topology analysis pinpointed key miRNAs-including osa-miR414, osa-miR1439, and osa-miR2919-as candidate topological hubs within the predicted network. Their predicted target genes, such as those encoding stress-responsive transcription factors and signaling proteins, suggest potential roles in coordinating post-transcriptional responses to combined stress. Network topology analysis pinpointed key miRNAs-including osa-miR414, osa-miR1439, and osa-miR2919-as candidate topological hubs within the predicted network. Their predicted target genes, such as those encoding stress-responsive transcription factors and signaling proteins, suggest potential roles in coordinating post-transcriptional responses to combined stress. Overall, this study provides a comprehensive map of circRNAs in rice under combined heat and drought stress, suggests their potential as ceRNAs based on predictive analysis, and lays a foundation for future experimental validation of circRNA-mediated regulation.

Oryza↗
PubMed1997

Escherichia coli RNA polymerase activity observed using atomic force microscopy.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Aluminum Silicates↗
PubMed2001

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids.

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH(2), where X = arginine, N(omega),N(omega)-dimethylarginine, DMA, or lysine) corresponding to residues 676-692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N(omega),N(omega)-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.

Amino Acid Sequence↗
PubMed2006

Structural studies of heterochiral DNA/DNA, RNA/RNA, and DNA/RNA duplexes.

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.

Circular Dichroism↗
PubMed2006

Restoration in vivo of defective hepatitis delta virus RNA genomes.

The 1679-nt single-stranded RNA genome of hepatitis delta virus (HDV) is circular in conformation. It is able to fold into an unbranched rodlike structure via intramolecular base-pairing. This RNA is replicated by host RNA polymerase II (Pol II). Such transcription is unique, because Pol II is known only for its ability to act on DNA templates. The present study addressed the ability of the HDV RNA replication to tolerate insertions of up to 1000 nt of non-HDV sequence either at an end of the rodlike RNA structure or at a site embedded within the rod. The insertions did not interfere with the ability of primary transcripts to be processed in vivo by ribozyme cleavage and ligation. The insertions greatly reduced the ability of genomes to replicate. However, when total RNA from such transfected cells was used to transfect new recipient cells, replicating HDV RNAs could be detected by Northern analyses. The size of the emerged RNAs was consistent with loss of the inserted sequences. RT-PCR, cloning, and sequencing showed that recovery involved removal of inserted sequences with or without small deletions of adjacent RNA sequences. Such restoration of the RNA genome is consistent with a model requiring intramolecular template-switching (RNA recombination) during RNA-directed transcription, in combination with biological selection for maintenance of the rodlike structure of the template.

Base Pairing↗
PubMed2006

Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses.

The linear, positive-stranded RNA genome of flaviviruses is thought to adopt a circularized conformation via interactions of short complementary sequence elements located within its terminal regions. This process of RNA cyclization is a crucial precondition for RNA replication. In the case of mosquito-borne flaviviruses, highly conserved cyclization sequences (CS) have been identified, and their functionality has been experimentally confirmed. Here, we provide an experimental identification of CS elements of tick-borne encephalitis virus (TBEV). These elements, termed 5'-CS-A and 3'-CS-A, are conserved among various tick-borne flaviviruses, but they are unrelated to the mosquito-borne CS elements and are located at different genomic positions. The 5'-CS-A element is situated upstream rather than downstream of the AUG start codon and, in contrast to mosquito-borne flaviviruses, it was found that the entire protein C coding region is not essential for TBEV replication. The complementary 3'-CS-A element is located within the bottom stem rather than upstream of the characteristic 3'-terminal stem-loop structure, implying that this part of the proposed structure cannot be formed when the genome is in its circularized conformation. Finally, we demonstrate that the CS-A elements can also mediate their function when the 5'-CS-A element is moved from its natural position to one corresponding to the mosquito-borne CS. The recognition of essential RNA elements and their differences between mosquito-borne and tick-borne flaviviruses has practical implications for the design of replicons in vaccine and vector development.

Animals↗
PubMed1992

Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.

Alternative Splicing↗