The Rpb9 subunit of RNA polymerase II binds transcription factor TFIIE and interferes with the SAGA and elongator histone acetyltransferases.

Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a beta-addition motif with the "jaw" of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable from rpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62-164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-Delta, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.     

Isolation and characterization of temperature-sensitive mutations in the gene (rpb3 ) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the α subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β′ subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, as in the case of the prokaryotic α₂β complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A–D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by over-expression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered.     

Amino acid substitution of the largest subunit of yeast RNA polymerase II: effect of a temperature-sensitive mutation related to G1 cell cycle arrest

A mammalian temperature-sensitive mutant tsAF8 shows cell cycle arrest at nonpermissive temperatures in mid-G1 phase. DNA sequence comparison of the largest subunit of RNA polymerase II (Rpb1) from the wild-type and the mutant shows that the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one rpb1 allele, giving rise to an Ala-to-Asp substitution at residue 315 in the protein. This amino acid substitution was introduced into the Schizosaccharomyces pombe rpb1 gene. Whereas tsAF8 cells showed growth defects and altered Rpb1 distribution at nonpermissive temperatures, yeast cells harboring this amino acid substitution did not show apparent temperature sensitivity. The effect of another temperature-sensitive Rpb1 mutation was also small. These results suggest that mutation of the rpb1 gene, which is critical in mammalian cells, may not be deleterious in yeast cells.     

Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP.LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP.LW.GKQ provokes a temperature-sensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Delta null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w. A ygr089-Delta null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.     

Isolation and characterization of temperature-sensitive mutations in the gene ( rpb3? ) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the α subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β′ subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, as in the case of the prokaryotic α₂β complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A–D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by over-expression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered. Received: 25 January 1999 / Accepted: 27 April 1999     

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth.

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.