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. 2002 Oct;22(20):6979-92.
doi: 10.1128/MCB.22.20.6979-6992.2002.

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach

Nevan J Krogan  1 , Minkyu Kim, Seong Hoon Ahn, Guoqing Zhong, Michael S Kobor, Gerard Cagney, Andrew Emili, Ali Shilatifard, Stephen Buratowski, Jack F Greenblatt
Affiliations

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach

Nevan J Krogan et al. Mol Cell Biol. 2002 Oct.

Abstract

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

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Figures

FIG. 1.
FIG. 1.
Isolation of protein complexes containing Spt4, Spt5, and Spt6. (A) TAPs of the Spt4/Spt5 complex were carried out on strains containing either no tagged protein or TAP-tagged versions of Spt4 or Spt5. Protein complexes were purified in the presence of 100 mM NaCl and were then analyzed by SDS-PAGE and silver staining. Spt4, Spt5, subunits of RNAPII, and a truncated form of Spt6 were identified by trypsin digestion and MALDI-TOF mass spectrometry. In the purification of tagged Spt5, the presence of Spt4 was identified by subjecting an aliquot of the eluate from the final column directly to trypsin digestion and tandem mass spectrometry. (B) TAP of Spt6. Purification using an extract containing a TAP-tagged version of Spt6 in 100 mM NaCl resulted in isolation of three forms of the tagged protein and a previously uncharacterized protein encoded by ORF YPR133c, whose gene product we have called Iws1 (interacts with Spt6). (C) TAP of Iws1. Tagging and isolation of Iws1 in 100 mM salt resulted in copurification of a stoichiometric amount of one form of Spt6. Because both Spt6-TAP and Iws1-TAP were run on the same gel, it could be concluded that Iws1 copurified with only the slowest-migrating form of Spt6.
FIG. 1.
FIG. 1.
Isolation of protein complexes containing Spt4, Spt5, and Spt6. (A) TAPs of the Spt4/Spt5 complex were carried out on strains containing either no tagged protein or TAP-tagged versions of Spt4 or Spt5. Protein complexes were purified in the presence of 100 mM NaCl and were then analyzed by SDS-PAGE and silver staining. Spt4, Spt5, subunits of RNAPII, and a truncated form of Spt6 were identified by trypsin digestion and MALDI-TOF mass spectrometry. In the purification of tagged Spt5, the presence of Spt4 was identified by subjecting an aliquot of the eluate from the final column directly to trypsin digestion and tandem mass spectrometry. (B) TAP of Spt6. Purification using an extract containing a TAP-tagged version of Spt6 in 100 mM NaCl resulted in isolation of three forms of the tagged protein and a previously uncharacterized protein encoded by ORF YPR133c, whose gene product we have called Iws1 (interacts with Spt6). (C) TAP of Iws1. Tagging and isolation of Iws1 in 100 mM salt resulted in copurification of a stoichiometric amount of one form of Spt6. Because both Spt6-TAP and Iws1-TAP were run on the same gel, it could be concluded that Iws1 copurified with only the slowest-migrating form of Spt6.
FIG. 2.
FIG. 2.
Detection of phosphorylation on putative CKII sites in Spt5, Spt6, and Iws1. (A) Identification of phosphorylation sites in Spt6. Tandem mass spectrometry was used to identify three tryptic fragments in the N-terminal region of Spt6 that were phosphorylated on serine residues (serines 95 [SEDD], 135 [SESD], and 207 [SDED]). Each phosphorylated serine residue in Spt6 was at the beginning of a consensus CKII site. (B) Phosphorylation of a conserved serine present in both Spt5 and Iws1. Mass spectrometry successfully detected a phosphorylation site on serine 89 (SDDD) of Iws1 as well as on serine 188 (SDDE) of Spt5; each of these occurs at the start of a putative CKII site. The acidic N-terminal regions of Spt5 and Iws1 have sequence similarity, and their phosphorylated serine residues align perfectly. The regions compared have 23% identity and 40% similarity.
FIG. 3.
FIG. 3.
Spt16/Pob3 associates with several protein complexes. (A) TAPs of Spt16 and Pob3 from TAP-tagged strains in buffers containing 150 mM NaCl. Purified preparations were analyzed by SDS-PAGE and silver staining. Spt16 and Pob3 were identified by mass spectrometry. (B) Purification of Spt16/Pob3 complexes in buffers containing 125 mM NaCl. Analysis by SDS-PAGE and silver staining revealed that a number of other polypeptides copurified with tagged Spt16 and Pob3. (C and D) Tagged Spt16 was purified in buffers containing 100 mM NaCl, resulting in an increased yield of proteins associated with Spt16/Pob3. The purified preparation was electrophoresed on gels containing 10% (C) or 15% (D) polyacrylamide. The additional proteins identified by mass spectrometry were Chd1, Ctr9, Paf1, Cdc73, Rtf1, Leo1, Yol054w, all four subunits of CKII, and histones. (E) TAP of tagged Chd1 in the presence of 100 mM salt. The purified protein was analyzed by SDS-PAGE, silver staining, and mass spectrometry. Tagged Chd1 copurified with seemingly stoichiometric amounts of the four subunits of CKII (CkaI, CkaII, CkbI, and CkbII) as well as substoichiometric amounts of Spt16 and Pob3. (F through H) TAPs of Leo1, Rtf1, and Ctr9 (F), Paf1 (G), and Cdc73 (H) (all in the presence of 100 mM NaCl) resulted in isolation of complexes containing Rtf1, Leo1, Ctr9, Paf1, and Cdc73, as well as Spt16 and Pob3, but no histones. (I) Sensitivities of deletion strains to 6-azauracil. Strains containing the rtf1Δ, paf1Δ, cdc73Δ, leo1Δ, chd1Δ, or dst1Δ deletion, as well as plasmid pRS316, were plated on SD-uracil medium with or without the drug 6-azauracil (50 μg/ml) and were grown at 30°C for 2 to 4 days. WT, wild type.
FIG. 3.
FIG. 3.
Spt16/Pob3 associates with several protein complexes. (A) TAPs of Spt16 and Pob3 from TAP-tagged strains in buffers containing 150 mM NaCl. Purified preparations were analyzed by SDS-PAGE and silver staining. Spt16 and Pob3 were identified by mass spectrometry. (B) Purification of Spt16/Pob3 complexes in buffers containing 125 mM NaCl. Analysis by SDS-PAGE and silver staining revealed that a number of other polypeptides copurified with tagged Spt16 and Pob3. (C and D) Tagged Spt16 was purified in buffers containing 100 mM NaCl, resulting in an increased yield of proteins associated with Spt16/Pob3. The purified preparation was electrophoresed on gels containing 10% (C) or 15% (D) polyacrylamide. The additional proteins identified by mass spectrometry were Chd1, Ctr9, Paf1, Cdc73, Rtf1, Leo1, Yol054w, all four subunits of CKII, and histones. (E) TAP of tagged Chd1 in the presence of 100 mM salt. The purified protein was analyzed by SDS-PAGE, silver staining, and mass spectrometry. Tagged Chd1 copurified with seemingly stoichiometric amounts of the four subunits of CKII (CkaI, CkaII, CkbI, and CkbII) as well as substoichiometric amounts of Spt16 and Pob3. (F through H) TAPs of Leo1, Rtf1, and Ctr9 (F), Paf1 (G), and Cdc73 (H) (all in the presence of 100 mM NaCl) resulted in isolation of complexes containing Rtf1, Leo1, Ctr9, Paf1, and Cdc73, as well as Spt16 and Pob3, but no histones. (I) Sensitivities of deletion strains to 6-azauracil. Strains containing the rtf1Δ, paf1Δ, cdc73Δ, leo1Δ, chd1Δ, or dst1Δ deletion, as well as plasmid pRS316, were plated on SD-uracil medium with or without the drug 6-azauracil (50 μg/ml) and were grown at 30°C for 2 to 4 days. WT, wild type.
FIG. 3.
FIG. 3.
Spt16/Pob3 associates with several protein complexes. (A) TAPs of Spt16 and Pob3 from TAP-tagged strains in buffers containing 150 mM NaCl. Purified preparations were analyzed by SDS-PAGE and silver staining. Spt16 and Pob3 were identified by mass spectrometry. (B) Purification of Spt16/Pob3 complexes in buffers containing 125 mM NaCl. Analysis by SDS-PAGE and silver staining revealed that a number of other polypeptides copurified with tagged Spt16 and Pob3. (C and D) Tagged Spt16 was purified in buffers containing 100 mM NaCl, resulting in an increased yield of proteins associated with Spt16/Pob3. The purified preparation was electrophoresed on gels containing 10% (C) or 15% (D) polyacrylamide. The additional proteins identified by mass spectrometry were Chd1, Ctr9, Paf1, Cdc73, Rtf1, Leo1, Yol054w, all four subunits of CKII, and histones. (E) TAP of tagged Chd1 in the presence of 100 mM salt. The purified protein was analyzed by SDS-PAGE, silver staining, and mass spectrometry. Tagged Chd1 copurified with seemingly stoichiometric amounts of the four subunits of CKII (CkaI, CkaII, CkbI, and CkbII) as well as substoichiometric amounts of Spt16 and Pob3. (F through H) TAPs of Leo1, Rtf1, and Ctr9 (F), Paf1 (G), and Cdc73 (H) (all in the presence of 100 mM NaCl) resulted in isolation of complexes containing Rtf1, Leo1, Ctr9, Paf1, and Cdc73, as well as Spt16 and Pob3, but no histones. (I) Sensitivities of deletion strains to 6-azauracil. Strains containing the rtf1Δ, paf1Δ, cdc73Δ, leo1Δ, chd1Δ, or dst1Δ deletion, as well as plasmid pRS316, were plated on SD-uracil medium with or without the drug 6-azauracil (50 μg/ml) and were grown at 30°C for 2 to 4 days. WT, wild type.
FIG. 3.
FIG. 3.
Spt16/Pob3 associates with several protein complexes. (A) TAPs of Spt16 and Pob3 from TAP-tagged strains in buffers containing 150 mM NaCl. Purified preparations were analyzed by SDS-PAGE and silver staining. Spt16 and Pob3 were identified by mass spectrometry. (B) Purification of Spt16/Pob3 complexes in buffers containing 125 mM NaCl. Analysis by SDS-PAGE and silver staining revealed that a number of other polypeptides copurified with tagged Spt16 and Pob3. (C and D) Tagged Spt16 was purified in buffers containing 100 mM NaCl, resulting in an increased yield of proteins associated with Spt16/Pob3. The purified preparation was electrophoresed on gels containing 10% (C) or 15% (D) polyacrylamide. The additional proteins identified by mass spectrometry were Chd1, Ctr9, Paf1, Cdc73, Rtf1, Leo1, Yol054w, all four subunits of CKII, and histones. (E) TAP of tagged Chd1 in the presence of 100 mM salt. The purified protein was analyzed by SDS-PAGE, silver staining, and mass spectrometry. Tagged Chd1 copurified with seemingly stoichiometric amounts of the four subunits of CKII (CkaI, CkaII, CkbI, and CkbII) as well as substoichiometric amounts of Spt16 and Pob3. (F through H) TAPs of Leo1, Rtf1, and Ctr9 (F), Paf1 (G), and Cdc73 (H) (all in the presence of 100 mM NaCl) resulted in isolation of complexes containing Rtf1, Leo1, Ctr9, Paf1, and Cdc73, as well as Spt16 and Pob3, but no histones. (I) Sensitivities of deletion strains to 6-azauracil. Strains containing the rtf1Δ, paf1Δ, cdc73Δ, leo1Δ, chd1Δ, or dst1Δ deletion, as well as plasmid pRS316, were plated on SD-uracil medium with or without the drug 6-azauracil (50 μg/ml) and were grown at 30°C for 2 to 4 days. WT, wild type.
FIG. 3.
FIG. 3.
Spt16/Pob3 associates with several protein complexes. (A) TAPs of Spt16 and Pob3 from TAP-tagged strains in buffers containing 150 mM NaCl. Purified preparations were analyzed by SDS-PAGE and silver staining. Spt16 and Pob3 were identified by mass spectrometry. (B) Purification of Spt16/Pob3 complexes in buffers containing 125 mM NaCl. Analysis by SDS-PAGE and silver staining revealed that a number of other polypeptides copurified with tagged Spt16 and Pob3. (C and D) Tagged Spt16 was purified in buffers containing 100 mM NaCl, resulting in an increased yield of proteins associated with Spt16/Pob3. The purified preparation was electrophoresed on gels containing 10% (C) or 15% (D) polyacrylamide. The additional proteins identified by mass spectrometry were Chd1, Ctr9, Paf1, Cdc73, Rtf1, Leo1, Yol054w, all four subunits of CKII, and histones. (E) TAP of tagged Chd1 in the presence of 100 mM salt. The purified protein was analyzed by SDS-PAGE, silver staining, and mass spectrometry. Tagged Chd1 copurified with seemingly stoichiometric amounts of the four subunits of CKII (CkaI, CkaII, CkbI, and CkbII) as well as substoichiometric amounts of Spt16 and Pob3. (F through H) TAPs of Leo1, Rtf1, and Ctr9 (F), Paf1 (G), and Cdc73 (H) (all in the presence of 100 mM NaCl) resulted in isolation of complexes containing Rtf1, Leo1, Ctr9, Paf1, and Cdc73, as well as Spt16 and Pob3, but no histones. (I) Sensitivities of deletion strains to 6-azauracil. Strains containing the rtf1Δ, paf1Δ, cdc73Δ, leo1Δ, chd1Δ, or dst1Δ deletion, as well as plasmid pRS316, were plated on SD-uracil medium with or without the drug 6-azauracil (50 μg/ml) and were grown at 30°C for 2 to 4 days. WT, wild type.
FIG. 4.
FIG. 4.
TAPs of TFIIF and TFIIS and detection of RNAPII interactions by Western blot analysis. (A) Purification of TFIIF by using TAP-tagged versions of Tfg1, Tfg2, and Tfg3. Purified proteins were analyzed by SDS-PAGE, silver staining, and mass spectrometry. Purification of any of the three subunits of TFIIF in buffers containing 150 mM NaCl results in isolation of all three components of TFIIF as well as a substoichiometric amount of RNAPII. (B) Tagging and purification of TFIIS. Purification of TFIIS (encoded by the DST1 gene) in buffers containing 100 mM NaCl resulted in the isolation of predominantly TFIIS. Further development of the silver stain revealed only background proteins and none that had copurified with TFIIS. (C) Western blot analysis of transcription elongation factors purified in the presence of 100 mM NaCl by using monoclonal antibody 8WG16, which binds to the CTD of the largest subunit of RNAPII, Rpb1. As a positive control, RNAPII was purified from a strain with a TAP tag on its third-largest subunit, Rpb3. Equal volumes of the column eluates from the various purifications were loaded onto the gel, except that approximately 10 times less of the Rpb3 purification was loaded. RNAPII, in the presence of 100 mM NaCl, copurified with TFIIF (Tfg1, -2, and -3), Spt4/Spt5, Spt6/Iws1, TFIIS, Spt16, and Paf1/Rtf1, but not with Elongator (Elp1 and Elp5) or Chd1.
FIG. 5.
FIG. 5.
ChIP assays with TFIIF, Paf1/Leo1/Ctr9/Cdc73/Rtf1, and Spt6/Iws1. ChIP was performed with strains containing TAP-tagged versions of the TFIIF subunits Tfg1, Tfg2, and Tfg3 (A), Paf1, Cdc73, Rtf1, Leo1, and Ctr9 (B), or Spt6 and Iws1 (C). To monitor the presence of each protein along the ADH1 gene, chromatin was immunoprecipitated with rabbit IgG-agarose and PCR amplified with primer pairs recognizing promoter (P), coding (C), and 3′ untranslated (U) regions (see schematic in panel A). Each PCR contained a second primer pair that amplified a region of chromosome V devoid of ORFs (marked by asterisks), thus providing an internal control for background. Input, signal from chromatin before immunoprecipitation. Primer pairs used are as follows: for P, ADH1-235 and ADH1-13; for C, ADH1844 and ADH11013; for U, ADH11231 and ADH11400; and for the nontranscribed region, Intergenic V-1 and Intergenic V-2.
FIG. 6.
FIG. 6.
Comprehensive interaction map for the RNAPII elongation factors. For details, see the text.
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