Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Abstract

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.

Fig 1. Alleles of RPA190 and RPA135 suppress the growth defect of the rpa49Δ mutant at various levels.

(A) The SGR1 mutant restores growth of the rpa49Δ mutant. Ten-fold serial dilutions of wild-type (WT), rpa49Δ single mutant, SGR1 single mutant, and SGR1/rpa49Δ double mutant strains were spotted on rich media to assess growth at 30°C and 25°C for three days. (B) Ten-fold dilutions of WT and rpa49Δ compared to rpa49Δ carrying various plasmids: pGL190_3 (RPA190-E1274K), pGL190_11 (RPA190-C1493R), pGL190_23 (RPA190-L1262P), pGL135_6prim (RPA135-R379G), pGL135_54 (RPA135-Y252H), or pGL135_33 (RPA135-F301S). Growth was evaluated after three days at 25°C. The strains and plasmids used are listed in S2 and S3 Tables, respectively.

Fig 2. Mapping of the modified residues in Rpa190, Rpa135 on the structure of Pol I and isolation of Rpa12 alleles.

(A) Two different views of the initially transcribing complex model and its 14 different subunits (PDB 5W66[15]). (B) Most mutated suppressor residues are clustered at the interface between the jaw (Rpa190, blue) and lobe (Rpa135, salmon) modules of Pol I. Note that residues 46–51 in Rpa12 (yellow) are part of this interface. (C) Zoom views of the areas containing the modified residues (Rpa190-N863, -S1259, -L1262, -E1274, -C1493; Rpa135-Y252, D299, S300, F301, R305; and Rpa12-S6, T49) shown in panel B. The figure was prepared with Pymol using the crystal structure of Pol I PDB 4C3I [4]). (D) Ten-fold dilutions of the rpa49Δ mutant carrying various plasmids: an empty pRS316 plasmid (-) YCp50-26 bearing RPA49 (RPA49), pRS316-A12-S6L (RPA12-S6L), or pRS316-A12-T49A (RPA12-T49A). Growth was evaluated after three days at 25°C or two days at 30°C.

Fig 3. RPA135-F301S and RPA12-S6L alleles restore growth and rRNA synthesis, and modulate Pol I occupancy of rDNA genes in the absence of Rpa49Ct.

(A) Doubling times of WT, rpa49ΔCt, RPA135-F301S, rpa49ΔCt/RPA135-F301S double mutant, RPA12-S6L, and the rpa49ΔCt/RPA12-S6L double mutant in a low rDNA copy number background (see S2 Table). (B) In vivo labelling of newly synthesized RNAs. WT (lane 1), rpa49ΔCt (lanes 2–4), RPA135-F301S (lane 5), the rpa49ΔCt/RPA135-F301S double mutant (lane 6), RPA12-S6L (Lane 7), the rpa49ΔCt/RPA12-S6L double mutant (lane 8) were grown to an OD₆₀₀ of 0.8. Cells were then pulse-labeled with [8-³H] adenine for 2 min. Samples were collected, and total RNA extracted and separated by gel electrophoresis. (C) Representative Miller’s spreads of WT, rpa49ΔCt, RPA135-F301S, rpa49ΔCt/RPA135-F301S, and rpa49ΔCt/RPA12-S6L double mutant. Panels on the right of each micrograph show interpretive tracing of the genes. Polymerases that appear on the gene are shown on the tracing by black dots. The number of polymerases counted on the genes is indicated below. N represents the number of individual spread genes used for quantification (see Materials and Methods). Scale bar = 200 nm. (D) ChIP analysis of Pol I occupancy at rDNA. Strains bearing WT Pol I, expressing Rpa49 lacking its C-terminal part (rpa49ΔCt), bearing suppressor mutation (RPA135-F301S) or double mutant (rpa49ΔCt/RPA135-F301S), were subjected to ChIP experiments using TAP tagged Rpa135, as described in Materials and Methods. Experiments were reproduced three times, representative Pol I occupancy relative to WT level is shown. Position of qPCR amplicons are depicted on rDNA unit. ** Marks significantly different values (p-values > 0.01) in student’s test of rpa49ΔCt with WT, RPA135-F301S and double rpa49ΔCt RPA135-F301S.

Fig 4. Rpa14, Rpa34, and the DNA mimicking loop of Rpa190 are not required for suppression.

Deletion of the DNA mimicking loop of Rpa190 (A) or of RPA34 (B) does not modulate the suppression activity of RPA135-F301S. (C) RPA135-F301S suppresses the synthetic lethality between rpa14Δ and rpa49Δ. Ten-fold serial dilutions were performed and growth evaluated after three days at 25°C.

Fig 5. RPA12 alleles can modulate the rpa49Δ-associated growth defect.

(A) Growth of the double mutants: rpa49Δ rpa12ΔCt, or rpa49Δ combined with full depletion of rpa12. Depletion of Rpa12 was achieved using a pGAL-RPA12 construct on glucose containing medium (strain OGT30-1c). Ten-fold serial dilutions of OGT30-1c bearing pRS316-A12 (WT), pRS316-A12-DCt expressing Rpa12 bearing a C-terminal deletion of residues 65–125 (rpa12ΔCt), or an empty plasmid pRS316 (-rpa12) were seeded onto media. The growth of rpa49Δ combined with RPA12 depletion was tested using strain OGT30-3c bearing pRS316-A12 (rpa49Δ), pRS316-A12-DCt (rpa12ΔCt rpa49Δ), or an empty plasmid pRS316 (-rpa12 rpa49Δ). Growth was assessed after four days at 25°C or 30°C. (B) The C terminus of Rpa12 is not required for suppression. Ten-fold serial dilutions of OGT30-1c (RPA49-WT), bearing pRS316-A12 (WT) or pRS316-A12-DCter (rpa12ΔCt), and OGT30-3c (rpa49Δ), bearing pRS316-A12 (WT), pRS316-A12-DCter (rpa12ΔCt), pRS316-A12-S6L (RPA12-S6L), or pTD10 (RPA12-S6L-ΔCt) were seeded onto media. Growth was assessed after four days at 25°C. (C) Suppression activity of RPA135-F301S is abolished in the absence of Rpa12. RPA12, under a regulatable promoter (pGAL) was either expressed (+RPA12; left panel) on galactose containing medium or repressed (-rpa12; right panel) on glucose containing medium. Ten-fold serial dilutions of RPA49, rpa49Δ, RPA49, RPA135-F301S, or rpa49Δ RPA135-F301S were grown at 25°C. Depletion of Rpa12 abolishes the suppression activity of RPA135-F301S (compare the left to the middle and right panels). Extended incubation (right panel, 10 days) was used to detect growth of the double mutant -RPA12 rpa49Δ on plates.

Fig 6. In vitro transcription assays of WT Pol I and Pol I mutants.

WT, Pol A* (lacking Rpa34 and Rpa49), Pol I bearing Rpa135-F301S, and Pol A* bearing Rpa135-F301S were affinity-purified and 5 nM of each enzyme was used in either promoter dependent (A) or tailed template assay (B). Promoter-dependent assays were performed in the presence of 70 nM Rrn3 and 20 nM CF. Radiolabelled transcripts were separated on a denaturing polyacrylamide/urea gel and detected using a PhosphorImager. Note that upper radiolabeled bands in the experiment analyzing promoter-dependent transcription are due to nonspecific background labelling. Experiments were reproduced at least three times, with different Pol I concentration; representative autoradiographes are shown.

Fig 7. RPA135-F301S led to over-production of rRNA in vivo.

(A) WT, RPA135-F301S, rrp6Δ and RPA135-F301SΔ rrp6Δ strains were grown to mid-log phase in glucose containing media. Cell samples were collected and total RNAs were extracted, separated by gel electrophoresis and transferred to a nylon membrane. The accumulation of the different (pre-) rRNAs was then analyzed by northern blot using different probes (see Materials and Methods). (B) In vivo labelling of newly-synthesized RNAs. WT, RPA135-F301S, rrp6Δ and RPA135-F301SΔ rrp6Δ strains were grown to an OD₆₀₀ of 0.8. Cells were then pulse-labeled with [8-³H] adenine for 40 seconds. Samples were collected, and total RNA extracted and separated by gel electrophoresis. Newly-synthetized RNA are revealed by autoradiography, loading control was performed by northern blot (PGK1 mRNA probe– 1831) on the same membrane. (C) High-resolution transcriptional run-on (TRO) analysis of WT, RPA135-F301S, rrp6Δ and RPA135-F301SΔ rrp6Δ strains. Nascent transcripts were labelled, and revealed using antisens oligonucleotides immobilized on slot-blot as described in Materials and Methods. Each experiment was performed twice; a representative example is shown in the lower left panel. NTS2, Pol I (mean of 5’ETS, 18S.2, 25S.1, 3’ ETS) are quantified relative to 5S signal in the lower right panel. Yeast rDNA unit is represented in the upper panel, with the position of the corresponding antisense oligonucleotides used.

Fig 8. Schematic representation of Pol I.

(A) Free monomeric Pol I with mobile Rpa49Ct and linker. (B) Initially transcribing complex (ITC) upon insertion of melted DNA in the presence of Rpa49 (purple). Rpa49Ct interacts with upstream DNA and the Rpa49-linker is folded, closing the cleft (black anchor). Movements of Rpa12 and the jaw with respect to the lobe are indicated with arrows. (C) Pol I lacking Rpa49 is likely defective in stabilizing the closed conformation in the DNA-binding cleft, resulting in a looser gripping of DNA inside the cleft (red asterisks). (D) Suppressor mutations (green) facilitate movement of the jaw/lobe interface and gripping of the DNA by the Pol I enzyme (red arrow), in the absence of Rpa49. (E) Combination of the presence of Rpa49 and a suppressor mutation (green dot) results in a super-active Pol I compared to the WT enzyme.