Cloning and sequence determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest subunit of RNA polymerase II.

The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined.     

Complete sequence of the gene encoding the largest subunit of RNA polymerase I of Trypanosoma brucei

We have set out to clone the trypanosomal gene encoding the largest subunit of RNA polymerase I. We screened a genomic library with a synthetic oligonucleotide probe encoding an eleven amino acid sequence motif, YNADFDGDEMN, which has been found in all eukaryotic RNA polymerase largest subunit genes analyzed so far. We isolated the Trp11 locus and determined the complete sequence of the gene encoded within this locus. The deduced amino acid sequence contains the highly conserved RNA polymerase domains as well as the previously identified RNA polymerase I-specific hydrophilic insertions. Therefore, the gene most closely resembles the largest subunit of RNA polymerase I.     

Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II

Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme.     

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.     

In trypanosomes the homolog of the largest subunit of RNA polymerase II is encoded by two genes and has a highly unusual C-terminal domain structure

We have isolated the genes encoding the largest subunit of all three classes of RNA polymerase from Trypanosoma brucei. While the pol II largest subunit is encoded by a single gene in all organisms examined to date, trypanosomes contain two copies of the gene. Both genes are expressed in the procyclic and bloodstream stages of the trypanosome life cycle. The two pol II genes differ from one another in their coding sequences by 21 silent substitutions and 4 amino acid substitutions. In the core part of the large subunit, the predicted polypeptides are similar to other eukaryotic RNA polymerases. Both trypanosome pol II polypeptides, like those of other eukaryotes, also have a unique C-terminal extension. However, this domain in the trypanosome polypeptides, unlike those of other eukaryotes, is not a tandemly repeated heptapeptide sequence.     

Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.     

Relatedness of archaebacterial RNA polymerase core subunits to their eubacterial and eukaryotic equivalents.

The sequence of the genes encoding the four largest subunits of the RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum was determined and putative translation signals were identified. The genes are more strongly homologous to eukaryotic than to eubacterial RNA polymerase genes. Analysis of the polypeptide sequences revealed colinearity of two pairs of adjacent archaebacterial genes encoding the B" and B' or A and C genes, respectively, with two eubacterial and two eukaryotic genes each encoding the two largest RNA polymerase subunits. This difference in sequence organization is discussed in terms of gene fusion in the course of evolution. The degree of conservation is much higher between the archaebacterial and the eukaryotic polypeptides than between the archaebacterial and the eubacterial enzyme. Putative functional domains were identified in two of the subunits of the archaebacterial enzyme.     

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.     

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.     

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.