Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I

We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I. The coding region comprises an open reading frame of 5151 bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194 kDa. Alignment of the deduced protein sequence reveals homology to the β′ subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases. However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding β′-like subunits of class II and III RNA polymerase. We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194. Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III. Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins. Received: 27 January 1997 / Accepted: 1 April 1997     

Isolation and characterization of the fission yeast gene rpa42 +, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42?kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.     

The fission yeast rpa17 + gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called α-related subunits which are shared only by polymerases I and III. The α-related subunits show limited homology with the α-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of α-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17?kDa which can functionally replace AC19 – an α-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae– and was thus named rpa17 ⁺. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17 ⁺ shows that the gene is essential for cell growth. Sequence comparison with other α-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the α-related subunits.     

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.     

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes. Received: 20 April 1999 / Accepted: 26 July 1999     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

The fission yeast rpa17+ gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called α-related subunits which are shared only by polymerases I and III. The α-related subunits show limited homology with the α-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of α-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17 kDa which can functionally replace AC19 – an α-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae– and was thus named rpa17 ⁺. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17 ⁺ shows that the gene is essential for cell growth. Sequence comparison with other α-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the α-related subunits. Received: 1 October 1998 / Accepted: 3 December 1998     

Isolation and characterization of cDNA encoding mouse RNA polymerase II subunit RPB14

By means of the yeast two-hybrid system using the 40-kDa subunit of mouse RNA polymerase I, mRPA40, as the bait, we isolated a mouse cDNA which encoded a protein with significant homology in amino acid sequence to the 12.5-kDa subunit of Saccharomyces cerevisiae RNA polymerase II, B12.5 (RPB11). Specific antibody raised against the recombinant protein that was derived from the cDNA reacted with a 14-kDa polypeptide in highly purified mammalian RNA polymerase II and did not react with any subunit of RNA polymerase I or III. Moreover, the antibody co-immunoprecipitated the largest subunit of mouse RNA polymerase II. These results provide biochemical evidence that the cDNA isolated, named mRPB14, encodes a specific subunit of RNA polymerase II, and indicate that the subunit organization of the enzyme is conserved between yeast and mouse. A possible role of the α-motif [Dequard-Chablat, M., Riva, M., Carles, C. and Sentenac, A., J. Biol. Chem. 266 (1991) 15300–15307] in the protein-protein interaction between mRPA40 and mRPB14 is also discussed.     

Regulation of RNA polymerase subunit synthesis in Escherichia coli: utilization of DNA-Intercalating drugs as a probe.

The effect of four DNA-intercalating drugs on the synthesis of the β and β′ subunits of Escherichia coli RNA polymerase was investigated. Acridine orange, proflavine, ethidium bromide, and berberine sulfate at sublethal doses caused a general reduction in cellular RNA and protein syntheses. Under this condition, acridine orange and proflavine rapidly led to overproduction of the β and β′ subunits in significant amounts. Ethidium bromide and berberine sulfate also caused overproduction of the two subunits but with a delay of 10 min at 30 °C. The β and β′ subunits of RNA polymerase became the major proteins being synthesized by E. coli cells after prolonged treatment with DNA-intercalating drugs. The level of the α subunit of RNA polymerase was not altered by any of the drugs tested. The overproduction of the β and β′ subunits induced by DNA-intercalating drugs is shown to require de novo synthesis of the ββ′ mRNA. These findings indicate that the expression of the ββ′ operon is regulated and that the synthesis of the α subunit is not regulated by the mechanism regulating the ββ′ operon. Taken together with evidence reported by others, these results strongly suggest that the concentration of intracellular free RNA polymerase plays a role in regulating the expression of the ββ′ operon.     

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.     

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.