The Paf1 complex physically and functionally associates with transcription elongation factors in vivo - PubMed (original) (raw)

The Paf1 complex physically and functionally associates with transcription elongation factors in vivo

Sharon L Squazzo et al. EMBO J. 2002.

Abstract

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.

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Figures

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Fig. 1. Rtf1–TAP co-purifies with Paf1, Cdc73, Ctr9, Leo1, Spt5 and Rpb1. Yeast strains expressing untagged and TAP-tagged forms of Rtf1 were processed in parallel as described in Materials and methods. (A) Silver-stained gel of purified proteins derived from ∼150 mg of protein extract. Identification of bands as Rtf1, Paf1 and Cdc73 was based upon immunoblot analyses carried out in parallel. Similar assignments were not made for Ctr9 and Leo1 (predicted masses of ∼125 and 54 kDa, respectively) because antibodies to these proteins were not available and because individual bands were not excised for our mass spectrometry analysis. (B) Immunoblots of purified proteins, visualized with antisera directed against Rtf1, Paf1, Cdc73 and Spt5. The monoclonal antibody 8WG16 was used to probe for Rpb1.

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Fig. 2. Rtf1 and Leo1 are members of the Paf1 complex. (A) Rtf1 co-immunoprecipitates with Leo1-HA1 and HA1-Paf1. Anti-HA1 immunoprecipitations were performed from extracts of strains lacking an HA1-tagged protein (mock, GHY1102), containing either HA1-tagged Paf1 (GHY1147) or Leo1 (GHY1141), and from a LEO1-HA1 paf1Δ strain (GHY1146). Proteins that precipitated with the HA1 antibody-bound beads were eluted with 1 M KCl (elution) and separated by SDS–PAGE. Proteins that remained bound to the beads after the 1 M KCl elution were liberated by boiling in sample buffer (beads). The HA1-tagged proteins and Rtf1 were visualized by immunoblotting. (B) Leo1-HA1 co-immunoprecipitates with Ctr9-Myc. Immunoprecipitations were performed on extracts of a Leo1-HA1 (mock, GHY1092) and a Ctr9-Myc Leo1-HA1 strain (GHY1184). (C) Rtf1 co- immunoprecipitates with Ctr9-Myc. Immunoprecipitations were performed on extracts of a strain lacking a myc tag (mock, GHY1002), a Ctr9-Myc strain (GHY1184) and a paf1Δ Ctr9-Myc strain (GHY1222). (D) Paf1, Leo1 and Rtf1 co-fractionate on a gel filtration column. Whole-cell extracts derived from a Leo1-HA1 strain (GHY1140) and a HA1-Paf1 strain (GHY1147) were fractionated on a Superose 6 column, and the indicated fractions from the eluates were analyzed by immunoblotting. Each experiment was performed at least twice, with peak elution of each protein reproducibly occurring in fraction 10.

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Fig. 3. Mutant phenotypes shared by Paf1 complex members. (A) Strains with the indicated genotypes and containing the his4-912δ and lys2-128δ insertions were grown on YPD media, replica-plated to minimal media supplemented with histidine, lysine, leucine, uracil and tryptophan (complete), and SC-his and SC-lys plates, and incubated for 3 days at 30°C. Strains: WT, FY118; rtf1Δ, KY522; paf1Δ, GHY917; cdc73Δ, GHY1083; leo1Δ, GHY240; ctr9Δ, GHY1157. (B) Strains with the indicated genotypes were grown on YPD media and then replica-plated to SC-ura media lacking or containing 50 µg/ml 6AU. Photographs were taken after 2 and 4 days of growth at 30°C. Strains: wild-type, KY286; rtf1Δ, KY425; paf1Δ, KY686; cdc73Δ, KY689; leo1Δ, KY690; ctr9Δ, KY695.

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Fig. 3. Mutant phenotypes shared by Paf1 complex members. (A) Strains with the indicated genotypes and containing the his4-912δ and lys2-128δ insertions were grown on YPD media, replica-plated to minimal media supplemented with histidine, lysine, leucine, uracil and tryptophan (complete), and SC-his and SC-lys plates, and incubated for 3 days at 30°C. Strains: WT, FY118; rtf1Δ, KY522; paf1Δ, GHY917; cdc73Δ, GHY1083; leo1Δ, GHY240; ctr9Δ, GHY1157. (B) Strains with the indicated genotypes were grown on YPD media and then replica-plated to SC-ura media lacking or containing 50 µg/ml 6AU. Photographs were taken after 2 and 4 days of growth at 30°C. Strains: wild-type, KY286; rtf1Δ, KY425; paf1Δ, KY686; cdc73Δ, KY689; leo1Δ, KY690; ctr9Δ, KY695.

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Fig. 4. rtf1Δ and paf1Δ strains are defective in PUR5 induction. Northern analysis of PUR5 transcription was performed on total RNA samples prepared from wild-type (KY589), rtf1Δ (KY453) and paf1Δ (KY687) cells. The strains were grown to ∼1 × 107 cells/ml in SC-ura media and then divided. 6AU was added to a final concentration of 75 µg/ml to one half of the culture and both cultures were grown at 30°C for the indicated times, in hours, prior to RNA isolation. Each lane contained 10 µg of total RNA. The same filters that were probed for PUR5 mRNA were stripped and probed for SED1 mRNA to serve as a loading control.

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Fig. 5. Suppression of the spt5Cs– growth defect by paf1 and leo1 mutations. Strains with the indicated genotypes were replica-plated to YPD media and incubated for 2 (30°C) or 4 (15°C) days. Strains: _spt5Cs_–, GHY92; wild type, FY653; _paf1-49 spt5Cs_–, GHY144; _leo1-43 spt5Cs_–, GHY141.

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Fig. 6. Transcription elongation factors associate with the Paf1 complex. (A) Spt5, Spt16 and Pob3 co-immunoprecipitate with HA1-Paf1 and Leo1-HA1. Anti-HA1 immunoprecipitations were performed from extracts of a strain lacking an HA1 epitope tag (mock, GHY1102), strains containing Leo1-HA1 (GHY1184), HA1-Paf1 (GHY1147) and from a paf1Δ Leo1-HA1 strain (GHY1146). The immunoprecipitates were analyzed by immunoblotting for Spt5, Spt16 and Pob3 as indicated. (B) Spt5 co-immunoprecipitates with Ctr9. Anti-Myc immunoprecipitations were performed on extracts of a strain lacking a Myc tag (mock, GHY1002), a Ctr9-Myc strain (GHY1184) and a paf1Δ Ctr9-Myc strain (GHY1222). Bound proteins were eluted from the anti-Myc beads by a 1 M salt elution (elution) and then by boiling the beads in sample buffer (beads). The immunoprecipitates were analyzed by immunoblotting for Spt5 and Ctr9, with an anti-Myc antibody, as indicated. (C) Whole-cell extracts were prepared from yeast strain FY1639 and used for immunoprecipitations of Rtf1. Western blots were probed with anti-Spt5, anti-Rtf1 and anti-TBP antisera. A 20 µg aliquot of whole-cell extract, 7 µg of unbound protein and three-fifths of the immunoprecipitated sample (beads) were analyzed by immunoblotting with the indicated antibodies. (D) Growth defects in paf1Δ dst1Δ and paf1Δ spt16-197 double mutants. Serial dilutions of cells with the indicated genotypes were spotted onto YPD media and grown for 2 days at 30°C. Strains: wild-type, FY653; paf1Δ, GHY917; dst1Δ, GHY285; spt16-197, GHY1059; paf1Δ dst1Δ, GHY1009; paf1Δ spt16-197, GHY1228.

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