The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts

Summary The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.     

The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail.

The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

RPC40, a unique gene for a subunit shared between yeast RNA polymerases A and C

Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.     

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34–40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30–40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria. Received: 5 January 2000 / Accepted: 7 March 2000     

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.     

RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III)

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.     

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.     

Molecular characterization and in silico analysis of RNA polymerase alpha subunit gene (rpoA) in Date Palm (Phoenix dactylifera L.) cv. Deglet Nour

The rpoA gene coding for the ??-subunit of DNA-dependent RNA polymerase located in the chloroplastic genome of date palm has been characterized from the elite cultivar Deglet Nour by cloning and sequencing of the PCR amplification product. The full length of rpoA-Pd (Phoenix dactylifera) gene was 1014 bp. The comparison in Genbank showed that the rpoA gene has a 100% homology with the Khalas cultivar of date palm and a strong homology with Oil Palm (99%). The deduced protein full length is 337 amino acid corresponding to 38,692 Da polypeptide. It contained an Alpha N-terminal domain (alpha-NTD) between 1 to 233 (aa) and Alpha C-terminal domain (alpha-CTD) between 266 to 337 (aa). Initially, we have compared the sequences of the full-length DNA rpoA gene from Date Palm and Oil palm, 15 mutations have been detected, 4 do not affect amino acid sequences. A multiple alignment of protein sequences of Date Palm and other plants shows 6 mutations specific for palms family and one is specific to monocots species. A multiple alignment of 35 nucleotide sequences from different plant species shows 3 SNPs specific to Date Palm, 6 SNPs specific to Palms family and 6 other to monocot species. The phylogenetic analysis performed in this work shows a strong similarity between Pd-rpoA and rpoA genes from other plant species, but it shows a great divergence with the rpoA of E. coli. To explain whether the separation of the two clades was due to selection pressure. We calculate the ratio Ka/Ks for different species. A synteny analyses of rpoA genes was effected, a high genomic synteny is observed for the ropA in all the species included in this study.     

The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5.

The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.     

Identification of the gene and the protein of RNA polymerase II subunit 9 (Rpb9) from the fission yeast Schizosaccharomyces pombe

Both the rpb9 gene and its cDNA encoding the subunit 9 of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. From the DNA sequences, Rpb9 was predicted to consist of 113 amino acid residues with a molecular mass of 13 175. S. pombe Rpb9 is 47, 40 and 36% identical in amino acid sequence to the corresponding subunits from Saccharomyces cerevisiae, human and Drosophila melanogaster, respectively. Previously, we failed to detect Rpb9 in the purified RNA polymerase II by amino-terminal micro-sequencing of proteolytic fragments of subunits separated by SDS-gel electrophoresis. After Western blot analysis using antibodies raised against the protein product of the newly isolated rpb9 gene, we found that the purified RNA polymerase II contains Rpb9.     

An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae.

The RPC34 gene of Saccharomyces cerevisiae was cloned by immunological screening, using antibodies raised against the C34 polypeptide of the RNA polymerase III (C). This single copy gene was located near the centromere of chromosome XIV. It included a coding sequence of 317 amino acids that strictly matched two internal oligopeptides of C34. This polypeptide is a specific component of RNA polymerase III, with no significant homology to any other RNA polymerase subunit known so far. It is an essential subunit, since inactivation by deletion or nonsense mutations led to a recessive lethal phenotype. Moreover, a partially blocked mutant, rpc34-F297, had a reduced tRNA synthesis in vivo but no detectable effect on 5 S RNA synthesis. The latter phenotype was observed for all conditionally defective RNA polymerase III mutants isolated so far.     

The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3.

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.