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.     

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.     

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.     

In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement.

Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in complexity, and many of their subunits display a high degree of sequence similarity to their eukaryotic counterparts. Here we describe specific protein-protein contacts present between individual recombinant RNAP subunits from the archaeon Methanococcus jannaschii. Subunits D and L interact specifically with each other in two-hybrid assays. D also interacts under the same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces cerevisiae, suggesting that essential elements of the binding surface between these proteins have been conserved across the archaeal/eukaryotic evolutionary domain boundary. Interactions between L and RPB3 or AC40 were, however, not detectable. Recombinant D and L subunits associate under in vitro conditions and copurify with each other during size-exclusion chromatography. Addition of an another recombinant subunit (N) to the D-L complex results in the formation of a triple complex. This D-L-N complex resembles the RPB3-RPB11-RPB10 or AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively. Our data provide evidence for a close similarity in the quaternary arrangement of a subset of archaeal and eukaryotic RNA polymerase subunits and the conservation of the protein-protein contacts formed between them.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

Arabidopsis thaliana RNA polymerase II subunits related to yeast and human RPB5

Arabidopsis thaliana contains at least four genes that are predicted to encode polypeptides related to the RPB5 subunit found in yeast and human RNA polymerase II. This subunit has been shown to be the largest subunit common to yeast RNA polymerases I, II, and III (RPABC27). More than one of these genes is expressed in Arabidopsis suspension culture cells, but only one of the encoded polypeptides is found in purified RNA polymerases II and III. This polypeptide has a predicted pI of 9.6, matches 14 of 16 amino acids in the amino terminus of cauliflower RPB5 that was microsequenced, and shows 42 and 53% amino acid sequence identity with the yeast and human RPB5 subunits, respectively.     

Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB9 was isolated and sequenced. RPB9 is a single copy gene on chromosome VII. The RPB9 sequence predicts a protein of 122 amino acids with a molecular mass of 14,200 Da. The yeast RPB9 subunit is similar in size and sequence to a protein encoded by DNA adjacent to the suppressor of the Hairy Wing gene in Drosophila melanogaster. Deletion of the RPB9 gene produced cells that were heat- and cold-sensitive. The RPB9 subunit, like the previously described RNA polymerase II subunit RPB4, is not essential for synthesis of mRNA, but is required for normal cell growth over a wide temperature range.     

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.     

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.     

Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity.     

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.     

Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology.

Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.     

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.