Properties of the Rpa49 subunit. (A) Homology between Rpa49 (S. cerevisiae) and its S. pombe (Rpa51) and human (PAF53) counterparts, shown by Blosum 62 matrices drawn at a 5/35 stringency. H. sapiens, Homo sapiens. (B) Two-hybrid screening with Gal4_BD-Rpa34. Six Gal4_AD-Rpa49 plasmids were isolated by genome-wide screening of a previously described two-hybrid library (17) by using Gal4_BD-Rpa34. Vertical lines denote the limits of the RPA49 inserts. β-Galactosidase (β-Gal) was tested as previously described (17). Data are shown for the Gal4_AD::RPA49(36,119) plasmid and for a reciprocal two-hybrid interaction between Gal4_BD-Rpa34 and Gal4_AD-Rpa49. The RPA49 open reading frame is shown with gray boxes representing four conserved parts of Rpa49, corresponding to positions 60 to 89, 128 to 154, 201 to 216, and 326 to 371 in S. cerevisiae (see Fig. S1 in the supplemental material). (C) Deletion mapping of the Rpa34-interacting domain on Rpa49. N-terminal and C-terminal deletion forms of Rpa49 were cloned as Gal4_BD fusions into the pVV213 vector (54) and tested against Gal4_BD-Rpa34 in a two-hybrid assay.

Association of Rpa34 and Rpa49 with Pol I in rpa34Δ or rpa49 mutants. ChIP assays were done on three independent cultures (comprising 100 ml of YPD) harvested at an A₆₀₀ of 0.6. Pol I was revealed by polyclonal anti-Rpa190 and anti-Rpa34 or anti-Rpa49 rabbit antibodies (49). DNA occupancy was defined by the ratio between the immunoprecipitation (IP) signal and the DNA input (IN) signal. The diagram at the bottom shows the approximate positions of the primers used for reverse transcription-PCR (RT-PCR) amplification. Arrows denote the start sites and transcription orientations of the 5S and 35S rRNA transcripts. NTS1 and NTS2, nontranscribed spacers 1 and 2; nt, nucleotides. (Left panels) Effect of rpa49 mutations on the binding of Rpa34 to Pol I. Strain D704-6C was transformed with pVV200 plasmids bearing the mutant (rpa49-119,416) and wild-type (WT) RPA49 alleles. (Right panels) Effect of rpa34Δ on the binding of Rpa49 in YPH499 (WT) and T4-1C (rpa34Δ).

Growth phenotypes of RNA Pol I mutants. (A) Sensitivity of RNA Pol I mutants to low temperature (25°C), mycophenolate (MPA), and 6-azauracil (AZA). Strains YPH499 (wild type [WT]), D704-6C (rpa49Δ), D360-1A (rpa14Δ), T4-1C (rpa34Δ), OG36-1A [rpa135(L656P)], SL46-1D [rpa135(D395N)], SL9-2C (rpa12Δ), and D101-CHIM2 (rpa43-chim2) were serially diluted and spotted onto YPD and onto synthetic complete medium lacking uracil but containing mycophenolate (10 μg/ml) or 6-azauracil (20 μg/ml). Plates were incubated for 3 days at 30°C. The box on the right summarizes the synthetic growth defects seen in rpa14Δ and top1Δ strains as monitored by plasmid shuffle tests (8) based on the counterselection of URA3 centromeric plasmids (with RPA12, RPA14, RPA34, or RPA49) on FOA medium. Lethality is indicated by a minus sign. Mutant strains that grew on FOA were streaked onto YPD for 3 days at 30°C. The + and (+) symbols denote a growth pattern identical or partially impaired relative to that of the corresponding wild-type control tested before the FOA chase. (B) Suppression of the caffeine sensitivity of the rpa34Δ mutant by RPA49 overexpression. The YPH499 wild type (WT), the isogenic mutant strain T4-1C (rpa34Δ), and T4-1C transformed with pGEN-A49 (rpa34Δ 2μ RPA49) were spotted onto YPD and onto YPD with caffeine at 1 g/liter and grown for 3 days at 30°C. (C) Suppression of rpa34Δ synthetically lethal defects by RPA49 overexpression. Strains OG1-L656P [rpa135(L656P) rpa34Δ], OG1-D395N [rpa135(D395N) rpa34Δ], and OG17-1C (top1Δ rpa34Δ) bearing the pOG1-A34 plasmid (2μm RPA34 URA3) were transformed with pGEN-A49 (2μm RPA49 TRP1) by using pGEN as an empty vector control (−). Suppression was detected by a plasmid shuffle assay on FOA, as described above. (D) Suppression of the rpa14Δ rpa34Δ synthetic lethality by RPA49 overexpression. Strains Y11277 (rpa34Δ::KanMX4) and D360-1A (rpa14Δ::HIS3) were crossed and transformed with pSLA49 (2μm RPA49 URA3), and meiotic tetrads were isolated by microdissection. The results presented show a tetratype with two parental and two recombinant segregants after replica plating onto 5-FOA, demonstrating that the rpa14Δ rpa34Δ strain (white frame) is unable to lose pSLA49. (E) Synthetic lethality of rpa49Δ with the RRN3::RPA43 fusion. Strains D781-10B [(RPA49 rpa43Δ)/2μm TRP1 RRN3::RPA43/CEN URA3 RPA43] and D780-6D [(rpa49Δ rpa43Δ)/2μm TRP1 RRN3::RPA43/CEN URA3 RPA43] were tested for their ability to lose the YCpA43-12 (CEN URA3 RPA43) plasmid by using a plasmid shuffle assay on FOA. The white frame around the D780-6D replica indicates the inability to lose YcpA43-12 in the rpa49Δ context.

Positions of the rpa135(D395N) and rpa135(L656P) mutations as deduced from the Pol II structure. Local sequence alignments are shown for the second-largest subunit of the three yeast RNA polymerases, for the corresponding archaeal subunits of Methanocaldococcus jannaschii (MjB′ and MjB″), and for the β subunit of Thermus aquaticus (Taq β). Two views of the Pol II spatial structure (Protein Data Bank coordinate 1WCM) are provided, showing the positions of Rpa12 (blue ribbons) and of the lobe and external folds. Asterisks indicate mutant positions.

Mutagenesis of RPA49. (A) Growth defects of rpa49 mutants. Strain D704-7C (rpa49Δ) was transformed with pVV200 plasmids (54) bearing the relevant wild-type sequences and rpa49 mutations corresponding to progressive N- and C-terminal deletions. Plasmid pVV214 was used as an empty vector control. Strain 49::HIS3 (see the tables in the supplemental material) was used to test the rpa49-338::HIS3 allele, in which the HIS3 insertion interrupts the Rpa49 coding sequence at H338 with a sequence encoding a GSAARSCSLACT extension beyond H338 (36). Four gray boxes represent conserved parts of Rpa49 (Fig. 1). Yeast cultures were serially diluted and spotted onto YPD (at 25 and 30°C and at 30°C with 1 g of caffeine [CAF]/liter) and synthetic complete medium with or without mycophenolate (MPA) at 20 μg/ml. Plates were incubated at 30°C for 3 days. The synthetic lethality of rpa49 mutations was tested in strains D699-13C (rpa14Δ rpa49Δ) and D684-2B (hmo1Δ rpa49Δ) bearing the pFB74 (RPA49 URA3) plasmid and transformed with the same plasmids indicated above. The resulting transformants were replica plated onto FOA medium to chase pFB74. Synthetically lethal effects were revealed by the lack of growth on FOA (8). In the case of rpa49-338::HIS3, synthetic lethality was deduced from meiotic crosses based on 12 tetrads. Lethality is indicated by a minus sign. (−) indicates very slow growth. The + and (+) symbols denote a growth pattern identical or partially impaired relative to that of the corresponding wild-type control. (B) Effect of rpa49 mutations on rDNA occupancy by Pol I. ChIP assays were done on D704-6C (rpa49Δ) transformed with the appropriate pVV200-derived plasmids. Pol I was revealed by polyclonal anti-Rpa190 rabbit antibodies (49). DNA occupancy was defined by the ratio between the immunoprecipitation (IP) signal and the DNA input (IN) signal. The diagram below shows the approximate positions of the primers used for RT-PCR amplification (see the tables in the supplemental material). Arrows denote the start sites and transcription orientations of the 5S and 35S rRNA transcripts. WT, wild type; NTS1 and NTS2, nontranscribed spacers 1 and 2.

Effect of RNA Pol I mutations on Rrn3-HA and Rrn7-HA occupancy. (A) Rrn3 occupancy in rpa43-chim2, rpa49Δ, and rpa49-338::HIS3 strains. ChIP assays were done on strain SL97-17C (rpa43Δ Rrn3-HA) bearing plasmid pGEN-A43 (wild type [WT]) or pGA43-chim2 (rpa43-chim2) and strains SL112-1C (rpa49Δ) and D612-5A (rpa49-338::HIS3) by using the same conditions described above. Rrn3-HA was revealed with anti-hemagglutinin A mouse antibody (12CA5). A schematic representation of the rpa43-chim2 strain is shown below. Numbers denote amino acid positions and correspond to the limits of the domains swapped between S. cerevisiae and S. pombe. IP, immunoprecipitation signal; IN, DNA input signal. (B) Rrn7 occupancy. ChIP assays were done on strains SL105-7A (rpa43Δ Rrn7-HA) and SL111-4A (rpa49Δ Rrn7-HA) bearing the same plasmids and under the same conditions described above.

Effect of mycophenolate (MPA) on wild-type (WT), rpa12Δ, and rpa43-chim2 strains. Cells were cultivated in SD+aa medium until they reached an A₆₀₀ of 0.2, treated (or left untreated) with 20 μg of mycophenolate/ml, and further grown for 30 min. In the case of Pol I, ChIP assays were done on strains SL97-17C [(rpa43::LEU2 Rrn3-HA)/YCPA43], used here as the wild-type control, SL9-2C (rpa12Δ), and D101-chim2 (rpa43-chim2). Pol I was revealed by polyclonal anti-Rpa190 rabbit antibodies (49). Five additional primers (5, 6, 7, 9, 10) were used for RT-PCR amplification and are listed in the tables in the supplemental material. The diagram at the bottom shows the approximate positions of the primers used for RT-PCR amplification (see the tables in the supplemental material). Arrows denote the start sites and transcription orientations of the 5S and 35S rRNA transcripts. SC, synthetic complete medium; IP, immunoprecipitation signal; IN, DNA input signal; NTS1 and NTS2, nontranscribed spacers 1 and 2.

Effect of mycophenolate (MPA) on rpa49Δ, rpa49-338::HIS3, and rpa135(L656P) mutants. (Left panels) Effect on Pol I (Rpa190) in wild-type (WT), rpa49Δ, rpa49-338::HIS3, and rpa135(L656P) strains. Cells were cultivated in SD+aa medium until they reached an A₆₀₀ of 0.2, treated (or left untreated) with 20 μg of mycophenolate/ml, and further grown for 30 min. ChIP assays were done on strains SL97-17C [(rpa43::LEU2 Rrn3-HA)/YCPA43], used here as the wild-type control, SL112-1C (Rrn3-HA rpa49Δ), SL107-3b (Rrn3-HA rpa49-338::HIS3), and OG36-1a [rpa135(L656P)]. Pol I was revealed by polyclonal anti-Rpa190 rabbit antibodies (49). (Right panels) Effect on Rrn3-HA. Cells were cultivated in SD+aa medium until they reached an A₆₀₀ of 0.2, treated (or left untreated) with 20 μg of mycophenolate/ml, and further grown for 30 min. ChIP assays were done on strains SL97-17C [(rpa43::LEU2 Rrn3-HA)/YCPA43], used here as the wild-type control, SL112-1C (Rrn3-HA rpa49Δ), SL107-3b (Rrn3-HA rpa49-338::HIS3), and D769-2C [Rrn3-HA rpa135(L656P)]. Rrn3-HA was revealed with anti-hemagglutinin A mouse antibody (12CA5). SC, synthetic complete medium; IP, immunoprecipitation signal; IN, DNA input signal.

Suppression of rpa49 mutations by an N-terminal deletion of Rpa43 (rpa43-35,326). (Upper panels) Strains D710-8A (rpa43Δ rpa49Δ) and SL107-3B (rpa43Δ rpa49-338::HIS3) bearing the YCP43-12 (URA3 RPA43) plasmid were transformed with plasmids pGEN-RPA43 (TRP1 RPA43) and pFB63 (TRP1 rpa43-35,326). Transformants were replica plated onto FOA medium to chase YCP43-12, tested on YPD (25°C) and synthetic complete medium with mycophenolate (MPA) at 0, 5, and 20 g/liter (30°C), and incubated for 3 days at 25°C, as described in the legend to Fig. 3. Strain YPH500 was used as a wild-type (WT) control. (Lower panels) ChIP assays were done on strains SL97-17C (WT) and SL107-3b (rpa49-338::HIS3) bearing plasmid pFB63 (rpa43-35,326) or pGEN-RPA43 (RPA43⁺) by using the same conditions described above, except that cells were cultivated in SD+aa medium and were harvested at an A₆₀₀ of 0.2. IP, immunoprecipitation signal; IN, DNA input signal.