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Ranajeet Ghose - One of the best experts on this subject based on the ideXlab platform.
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Cystoviral RNA-directed RNA Polymerases: Regulation of RNA synthesis on multiple time and length scales.
Virus research, 2017Co-Authors: Sébastien Alphonse, Ranajeet GhoseAbstract:P2, an RNA-directed RNA Polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.
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Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-directed RNA Polymerase.
Biochemistry, 2015Co-Authors: Sébastien Alphonse, Haiyan Wang, Shibani Bhattacharya, Ranajeet GhoseAbstract:Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA Polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA Polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.
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cystoviral Polymerase complex protein p7 uses its acidic c terminal tail to regulate the RNA directed RNA Polymerase p2
Journal of Molecular Biology, 2014Co-Authors: Sébastien Alphonse, Ranajeet Ghose, Shibani Bhattacharya, Jamie J Arnold, Hsin Wang, Brian Kloss, Craig E CameronAbstract:In bacteriophages of the cystovirus family, the Polymerase complex (PX) encodes a 75-kDa RNA-directed RNA Polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former toward template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses.
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Structure of the RNA-directed RNA Polymerase from the Cystovirus φ12
Proteins, 2013Co-Authors: Zhen Ren, Matthew C. Franklin, Ranajeet GhoseAbstract:We have determined the structure of P2, the self-priming RdRp from cystovirus ϕ12 in two crystal forms (A, B) at resolutions of 1.7 A and 2.1 A. Form A contains Mg2+ bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts-mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo. Proteins 2013; 81:1479–1484. © 2013 Wiley Periodicals, Inc.
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Slow Conformational Dynamics in the Cystoviral RNA-directed RNA Polymerase P2 – Influence of Substrate Nucleotides and Template RNA
Biochemistry, 2011Co-Authors: Zhen Ren, Ranajeet GhoseAbstract:The RNA-directed RNA Polymerase P2 from cystovirus ϕ6 catalyzes the de novo synthesis of positive and negative strands of the viral double-stranded RNA genome. P2 is mobile on the slow, microsecond to millisecond time scale with various motional modes, putatively assisting in RNA translocation and catalysis. Here we investigate the influence of the extreme 3′-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on these motional modes using multiple-quantum NMR spectroscopy. We find that P2, in the presence of templates bearing the proper genomic 3′-ends or the preferred initiation nucleotide, displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.
Drake C. Stenger - One of the best experts on this subject based on the ideXlab platform.
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Reovirus genomes from plant-feeding insects represent a newly discovered lineage within the family Reoviridae
Virus Research, 2011Co-Authors: Allyn Spear, Mark S. Sisterson, Drake C. StengerAbstract:Abstract A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5′-AGAGA and CGAUGUUGU-3′) of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA Polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species.
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plant feeding insects harbor double stranded RNA viruses encoding a novel proline alanine rich protein and a Polymerase distantly related to that of fungal viruses
Virology, 2010Co-Authors: Allyn Spear, Mark S. Sisterson, R K Yokomi, Drake C. StengerAbstract:Abstract Novel double-stranded RNAs (∼ 8 kbp) were isolated from threecornered alfalfa hopper ( Spissistilus festinus ) and beet leafhopper ( Circulifer tenellus ), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA Polymerase (RdRp). Expression of the 3’-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.
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plant feeding insects harbor double stranded RNA viruses encoding a novel proline alanine rich protein and a Polymerase distantly related to that of fungal viruses
Virology, 2010Co-Authors: Allyn Spear, Mark S. Sisterson, R K Yokomi, Drake C. StengerAbstract:Novel double-stranded RNAs (approximately 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA Polymerase (RdRp). Expression of the 3'-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.
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A new Phytoreovirus infecting the glassy-winged sharpshooter (Homalodisca vitripennis).
Virology, 2009Co-Authors: Drake C. Stenger, Mark S. Sisterson, Rodrigo Krugner, Elaine A. Backus, Wayne B. HunterAbstract:A new virus species of the genus Phytoreovirus was isolated from glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar (Hemiptera: Cicadellidae), in California and designated here as Homalodisca vitripennis reovirus (HoVRV). Extraction of nucleic acid from GWSS adults collected from three Californian populations revealed an array of double-stranded (ds) RNA species that was soluble in 2 M LiCl and resistant to degradation upon exposure to S1 nuclease and DNase. Analysis of nucleic acid samples from single GWSS adults indicated that HoVRV dsRNA accumulated to high titer in individual insects. Double-shelled isometric virus particles purified from GWSS adults resembled those observed in thin sections of GWSS salivary glands by transmission electron microscopy. Purified HoVRV virions contained 12 dsRNA segments that, based on complete nucleotide sequences, ranged in size from 4475 to 1040 bp. Sequence comparisons indicated that the HoVRV dsRNA segments were most closely related (58.5 to 43.7% nt sequence identity) to the corresponding genome segments of Rice dwarf virus (RDV). Each HoVRV dsRNA segment encoded a single open reading frame (>300 nts) except for segment 11, which appears to be dicistronic. Terminal nucleotide sequences of HoVRV positive-sense RNAs were similar to other phytoreoviruses (GGCG or GGCA at the 5'-end and UGAU or CGAU at the 3'-end) with adjacent imperfect inverted repeats potentially able to base pair. Phylogenetic analyses of the RNA-directed RNA Polymerase (encoded by segment 1) and the outer capsid protein (encoded by segment 8) confirmed placement of HoVRV as a species of the genus Phytoreovirus sharing a most recent common ancestor with RDV. Reverse transcription-Polymerase chain reaction assays revealed that HoVRV infection of GWSS in California was common and that the virus also occurred in GWSS populations from the Carolinas and Texas.
Patrick K. T. Shiu - One of the best experts on this subject based on the ideXlab platform.
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Complex Formation of RNA Silencing Proteins in the Perinuclear Region of Neurospora crassa
Genetics, 2015Co-Authors: Logan M. Decker, Thomas M. Hammond, Hua Xiao, Erin C. Boone, Benjamin S. Shanker, Shannon F. Boone, Shanika L. Kingston, Seung A. Lee, Patrick K. T. ShiuAbstract:In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA Polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.
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SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery
G3 (Bethesda Md.), 2011Co-Authors: Thomas M. Hammond, Hua Xiao, Erin C. Boone, Tony D. Perdue, Patricia J. Pukkila, Patrick K. T. ShiuAbstract:In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA Polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species.
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Neurospora spore killers Sk-2 and Sk-3 suppress meiotic silencing by unpaired DNA.
Genetics, 2007Co-Authors: Namboori B. Raju, Robert L. Metzenberg, Patrick K. T. ShiuAbstract:In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA Polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer × sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer × sensitive crosses. We suggest that the region contains a suppressor of MSUD.
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SAD-2 is required for meiotic silencing by unpaired DNA and perinuclear localization of SAD-1 RNA-directed RNA Polymerase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Patrick K. T. Shiu, Denise Zickler, Namboori B. Raju, Gwenaël Ruprich-robert, Robert L. MetzenbergAbstract:A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA Polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2. Mutated Sad-2 (RIP) alleles, like those of Sad-1, are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2(+) gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.
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Meiotic Silencing by Unpaired DNA
Cell, 2001Co-Authors: Patrick K. T. Shiu, Denise Zickler, Namboori B. Raju, Robert L. MetzenbergAbstract:Abstract The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora . The wild-type allele, sad-1 + , encodes a putative RNA-directed RNA Polymerase.
Heinz L Sanger - One of the best experts on this subject based on the ideXlab platform.
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Isolation of an RNA-directed RNA Polymerase–Specific cDNA Clone from Tomato
The Plant cell, 1998Co-Authors: Winfried Schiebel, Heinz L Sanger, Thierry Pélissier, L Riedel, S Thalmeir, R Schiebel, D Kempe, F Lottspeich, M WasseneggerAbstract:A 3600-bp RNA-directed RNA Polymerase (RdRP)–specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)–, and poly(U)–Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using Polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA Polymerase in homology-dependent gene silencing is discussed.
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RNA directed RNA Polymerase from tomato leaves i purification and physical properties
Journal of Biological Chemistry, 1993Co-Authors: Winfried Schiebel, Bernd Haas, S Marinkovic, A Klanner, Heinz L SangerAbstract:Abstract An RNA-directed RNA Polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
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RNA-directed RNA Polymerase from tomato leaves. II. Catalytic in vitro properties.
The Journal of biological chemistry, 1993Co-Authors: Winfried Schiebel, Bernd Haas, A Klanner, S Marinković, Heinz L SangerAbstract:The catalytic properties of electrophoretically homogeneous RNA-directed RNA Polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.
Winfried Schiebel - One of the best experts on this subject based on the ideXlab platform.
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Isolation of an RNA-directed RNA Polymerase–Specific cDNA Clone from Tomato
The Plant cell, 1998Co-Authors: Winfried Schiebel, Heinz L Sanger, Thierry Pélissier, L Riedel, S Thalmeir, R Schiebel, D Kempe, F Lottspeich, M WasseneggerAbstract:A 3600-bp RNA-directed RNA Polymerase (RdRP)–specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)–, and poly(U)–Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using Polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA Polymerase in homology-dependent gene silencing is discussed.
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RNA directed RNA Polymerase from tomato leaves i purification and physical properties
Journal of Biological Chemistry, 1993Co-Authors: Winfried Schiebel, Bernd Haas, S Marinkovic, A Klanner, Heinz L SangerAbstract:Abstract An RNA-directed RNA Polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
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RNA-directed RNA Polymerase from tomato leaves. II. Catalytic in vitro properties.
The Journal of biological chemistry, 1993Co-Authors: Winfried Schiebel, Bernd Haas, A Klanner, S Marinković, Heinz L SangerAbstract:The catalytic properties of electrophoretically homogeneous RNA-directed RNA Polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.
