Dataset Information

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer.

ABSTRACT: The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.

SUBMITTER: Watanabe T

PROVIDER: S-EPMC104018 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer.

Watanabe T T Honda A A Iwata A A Ueda S S Hibi T T Ishihama A A

Journal of virology 19990401 4

The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine ta ...[more]

PMID: 10074108

Similar Datasets

Project description:All sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, ?1 and ?2. In all other bacteria studied to date, a dimer of identical ? subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition ?2??'). Based on an observation that both ?1 and ?2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell.By in vitro assembly from fully denatured state, we determined that both Francisella ? subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the ?1 and ?2 N-terminal domains (NTDs, responsible for dimerization). NTDs of ?2 did not interact detectably, while weak interaction between ?1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (?', ?) and ?1. No activity was observed with RNAP reconstitution reactions containing ?2, while robust transcription activity was detected in reactions containing ?1 and ?2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within ?-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.The results strongly suggest that most Francisella RNAP contains ? heterodimer with a minor subfraction possibly containing ?1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, ?1, interacts with the ? subunit during the ?2? RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.

Project description:The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.

Dataset Information

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer.

Publications

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets