Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae.

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14. 5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae. Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits. We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly. We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.     

Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe.

The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase. To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S. pombe by quantitative immunoblotting. Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of beta', beta and alpha subunits, respectively, of prokaryotic RNA polymerase. On the other hand, the levels of small-sized subunits were between 2- to 15-fold higher than these three core subunits. The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II-specific core subunits. The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S. pombe whole cell extracts. The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms. Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S. pombe cell was estimated to be about 10,000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)-fused Rpb proteins. In agreement with the biochemical analysis, the GFP-Rpb1 and GFP-Rpb3 fusions were present in the nuclei but the GFP-Rpb4 was detected in the cytoplasm as well as the nuclei.     

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.     

Functional conservation of RNA polymerase II in fission and budding yeasts

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits.     

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.     

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.     

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.     

Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast

[Rpb1 and Rpb2] Mapping of the contact sites␣on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the β' subunit of prokaryotic RNA polymerases. In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5. On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the β subunit of prokaryotic RNA polymerases. Possible functions of the regions H in the subunits 1 and 2 are discussed. Received: 10 December 1997 / Accepted: 14 April 1998