Similar upstream regulatory elements of genes that encode the two largest subunits of RNA polymerase II in Saccharomyces cerevisiae.

We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae. A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes. Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression. A T-rich region downstream of these sites was also important for expression. Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites. The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p. The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately.     

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.     

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.     

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.     

An attempt to unify the structure of polymerases

With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.