Six active phage-type RNA polymerase genes in Nicotiana tabacum

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.     

Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.     

One RNA polymerase serving two genomes

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.     

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

   

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.     

Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

Background  

The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation.     

A combined in vitro / in vivo selection for polymerases with novel promoter specificities

Background  

The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro / in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10³ – 10⁶) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendent mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.     

Evolution of Phage-Type RNA Polymerases in Higher Plants: Characterization of the Single Phage-Type RNA Polymerase Gene from <Emphasis Type="Italic">Selaginella moellendorffii</Emphasis>

Selaginella moellendorfii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases (RpoTs) were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3006 nt, encoding a putative protein of 1002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. In phylogenetic analyses, the Selaginella RpoT polymerase is in a sister position to all other phage-type polymerases of angiosperms. However, according to its conserved exon–intron structure, the Selaginella RpoT gene is representative of the molecular evolutionary lineage giving rise to the RpoT gene family of flowering plants. The N-terminal transit peptide of SmRpoT is shown to confer targeting of green fluorescent protein exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. Angiosperms and the moss P. patens possess small gene families encoding RpoTs, which include mitochondrial- and chloroplast-targeted RNA polymerases. In striking contrast, the Selaginella RpoT gene is shown to be single-copy, although Selaginella, as a lycophyte, has a phylogenetic position between Physcomitrella and angiosperms. Thus, there is no evidence that Selaginella may contain a nuclear-encoded phage-type chloroplast RNA polymerase.     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Isolation of RNA polymerases from the water mold Achlya

The DNA-dependent RNA polymerases I, II, and III (ribonucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) from Achlya ambisexualis E87 (male), have been isolated. The highly purified RNA polymerase I was found to be composed of polypeptides with the following molecular weights (·10^-4): 18.5, 14, 11.8, 7.3, 6.1, 4.9, 4.4, 2.8. RNA polymerase II showed a 400-fold higher resistance against -amanitin than mammalian or higher plant RNA polymerase II.     

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

Background  

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. Pseudomonas putida GPo1 and many other Pseudomonas species contain two different class II polymerases, encoded by phaC1 and phaC2. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.     

Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris

A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes. We have designated this protein NsRpoTp.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.