Spt8 and Med13 are TFIIS partners in a two-hybrid assay. (A) Spatial structure of the RNA polymerase II–TFIIS complex domain I, domain II, the inter-domain linker and domain III of TFIIS are shown in grey, blue, cyan and brown, respectively. The borders of each domain are taken from the crystal structure recently reported by Kettenberger et al (2003). The structure of the N-terminal domain I (positions 1–111, symbolised here by a dashed line) was solved in solution by nuclear magnetic resonance (Booth et al, 2000), but was not determined in association with Pol II. The Rpb4 and Rpb9 subunits of Pol II are indicated in red and green, respectively. The black sphere locates the catalytic Mg²⁺ A. This figure was prepared with the RASMOL software (www.umass.edu/microbio/rasmol/). (B) General organisation of Spt8, Med13 and TFIIS. TFIIS: The TFIIS domains are shown in the same code colour as in (A). A horizontal thick line denotes the minimal region supporting a two-hybrid interaction with Spt8 and Med13, based on the data shown in (C). Spt8: Stars and black boxes indicate WD40-like domains and acidic stretches, respectively. The horizontal thick line denotes the TFIIS-binding region (positions 364–465) as defined by the smallest domain common to the 25 pACT2-SPT8 clones identified by a two-hybrid screening using pVV70 as bait vector (Table II). An example of two-hybrid interaction is shown in (C). Med13: The stripped box corresponds to a region with strong homology between fungal forms of Med13. The horizontal thick line denotes the TFIIS-binding region (positions 320–514), as defined by the smallest domain common to the 11 pACT2-MED13 clones selected by two-hybrid screening. An example of two-hybrid interaction is shown in (C). (C) Two-hybrid interactions with TFIIS. Left panel: The complete coding sequence of TFIIS and various N-terminal or C-terminal fragments thereof were fused to the C-end of the GAL4_BD(1–147) DNA-binding domain (plasmids pVV70–pVV75, Table II) and tested for their interaction with Spt8 (plasmid pSPT8-84) and Med13 (plasmid pMED13-111). Transformants were obtained in strain Y190 and tested at 30°C for β-galactosidase activity in an overlay assay (Flores et al, 1999). Right panel: β-Galactosidase activity was assayed as described by Miller (1972). The average values and their standard deviation were calculated from assays performed on three independent transformants, using the same plasmid combination as in the left panel. (D) Conservation of the TFIIS N-terminal domain. The S. cerevisiae (Sc) sequence of TFIIS was compared to the current human genome. Homology search was made using the Psi-blast algorithm and improved by manual inspection. The number in brackets indicates the length of each polypeptide. The two last sequences (accession number AAH35374 and XP294568) correspond to putative gene products that are related to TFIIS but lack the invariant RSADE motif.

Co-purification of TFIIS with Med13 and Cdk8 in cell-free extracts. (A) Co-purification of TFIIS∷3HA with immunoprecipitated Med13∷13Myc. Strains YPH499 (SPT8⁺) and YBV61 (spt8Δ) were transformed with plasmids encoding the Med13∷13Myc fusion (pVV226) and/or the TFIIS∷3HA fusion (pVV227). Protein crude extracts were prepared as described (Van Mullem et al, 2002b). Med13∷13Myc was immunoprecipitated by mouse monoclonal anti-Myc antibodies. Proteins (IP) were separated by SDS–PAGE, along with 10 μg of crude extracts, and revealed by Western blotting with monoclonal anti-Myc or anti-HA antibodies. The band noted IgG corresponds to the heavy chains of the mouse anti-Myc antibodies used for the immunopurification. (B) Co-purification of Med13∷3HA with immunoprecipitated TFIIS∷13Myc. Strain YPH499 was transformed with plasmids encoding the TFIIS∷13Myc fusion (pVV234) and/or the Med13∷3HA fusion (pVV229). Protein extraction, immunopurification and detection were as above. (C) Co-purification of Cdk8∷13Myc with immunoprecipitated TFIIS∷3HA. Strain YPH499 was transformed with plasmids encoding the TFIIS∷3HA fusion (pVV228) and/or the Cdk8∷13Myc (pVV233). TFIIS∷3HA was immunoprecipitated by mouse monoclonal anti-HA antibodies. Protein extraction and detection were as above. (D) Co-purification of TFIIS∷3HA with immunoprecipitated Cdk8∷13Myc. Strains YPH499 (MED13⁺) and YBV39 (med13Δ) were transformed with plasmids encoding the Cdk8∷13Myc (pVV233) and/or the TFIIS∷3HA fusion (pVV228). Protein extraction, immunopurification and detection were as in (A, B). No co-purification was observed when Cdk8∷13Myc and TFIIS∷3HA were expressed from their respective chromosomal locus (data not shown).

Co-purification of TFIIS with components of SAGA in cell-free extracts. (A) Co-purification of TFIIS∷3HA with immunoprecipitated Spt8∷13Myc. Strains YPH499 (MED13⁺) and YBV39 (med13Δ) were transformed with plasmids encoding the Spt8∷13Myc fusion (pVV225) and/or the TFIIS∷3HA fusion (pVV227). Protein extraction, immunopurification and detection were as for Figure 2A. (B) Co-purification of Spt8∷3HA with immunoprecipitated TFIIS∷13Myc. Strain YPH499 was transformed with plasmids encoding the TFIIS∷13Myc fusion (pVV234) and/or the Spt8∷3HA fusion (pVV230). Protein extraction, immunopurification and detection were as in Figure 2A. (C) Co-purification of Gcn5∷13Myc and Spt7∷13Myc with immunoprecipitated TFIIS∷3HA. Strain YPH499 was transformed with plasmids encoding the TFIIS∷3HA fusion (pVV228) and/or the Gcn5∷13Myc (pVV231) or the Spt7∷13Myc (pVV232) fusions. Protein extraction, immunopurification and detection were as for Figure 2C. (D) Co-purification of TFIIS∷3HA with immunoprecipitated Gcn5∷13Myc and Spt7∷13Myc. Strains YPH499 (SPT8⁺) and YMW220 (spt8Δ) were transformed with plasmids encoding the Gcn5∷13Myc (pVV231) or Spt7∷13Myc (pVV232) fusions and/or the TFIIS∷3HA fusion (pVV228). Protein extraction, immunopurification and detection were as for Figure 2A. No co-purification was observed when Gcn5∷13Myc and TFIIS∷3HA were expressed from their respective chromosomal locus (data not shown).

Phenotypic effects of dst1Δ and spt8Δ mutants. (A) Mycophenolate sensitivity and epistasis with rpb9Δ. Wild type (YPH499), dst1Δ (CMKy1), spt7Δ (Y03218), spt3Δ (Y04228), spt8Δ (Y12666), rpb9Δ (YVV9), rpb9Δ dst1Δ (YVV62), dst1Δ spt8Δ (YBV55) and rpb9Δ spt8Δ (YMW238) cells were spotted on SC medium without or with mycophenolate (1, 5, 10 or 50 μg/ml). Plates were incubated at 25°C for 5 days. (B) Recapitulation of synthetic phenotypes involving dst1Δ and spt8Δ. Symbols: +, wild type; (+), partial growth defect at 30°C; −, lethality; ts, temperature sensitivity. In each case, synthetic lethal patterns were based on the analysis of at least 15 meiotic tetrads. Stars indicate that spt7Δ dst1Δ or spt7Δ spt8Δ mutants can be temperature sensitive depending on the genetic background. This phenotype probably reflects the intervention of a third mutation present in some genetic backgrounds, but the corresponding gene has not been identified. (C) Caffeine sensitivity of dst1 mutants. CMKy1 (dst1Δ) was transformed with pCM-DST1 (DST1), pCM-ΔN (dst1-133,309) and the empty vector pCM185. Transformants were streaked on YPD with 10 mM caffeine and incubated for 7 days at 30°C, using YPH500 (WT) as a wild-type control. (D) Mycophenolate sensitivity of dst1 mutants. Wild type (DY236-6B) and CMKy1 (dst1Δ) were transformed with pVV80 (DST1) and pVV81 (dst1-133,309), using the empty vector (pGEN) as a control. Transformants were spotted on SC-W and SC-W with 50 μg/ml of mycophenolate. Plates were incubated at 25°C for 3 days.

Spt8 and the N-terminal domain of TFIIS are critical in an rpb4Δcontext. (A) Synthetic lethality between spt8Δ and rpb4Δ. Left panel: Strains ESH13-8D (spt8Δ) and ESH27-15C (rpb4Δ) were crossed and submitted to tetrad analysis. Plates were incubated on YPD. Minute colonies corresponding to spt8Δ rpb4Δ double mutants were obtained but could not be further propagated on YPD. One tetratype is shown after 5 days at 30°C. Right panel: The cross above was repeated but diploid strains were transformed with pYX212-RPB4 (2 μURA3 RPB4) prior to sporulation. Strain ESH28-2A (spt8Δ rpb4Δ/2 μ URA3 RPB4) was isolated in the meiotic offspring and then transferred on FOA to counterselect the pYX212-RPB4 plasmid. This material was then transferred to YPD and incubated for 5 days at 30°C and compared to the strain ESH28-2A as a control. (B) Synthetic lethality between rpb4Δ and mutants lacking the N-terminal domain of TFIIS. Left panel: Strain ESH1 (dst1Δ) was crossed to SL21-3A (rpb4Δ) and submitted to tetrad analysis. Plates were incubated on YPD. Minute colonies corresponding to dst1Δ rpb4Δ double mutants were obtained but have an extremely poor viability when further propagated on YPD. One tetratype is shown after 5 days at 30°C. Right panel: Strain ESH29-1B (dst1Δ rpb4Δ with the 2 μ URA3 RPB4 plasmid pYX212-RPB4) was transformed by the TRP1 centromeric plasmids pCM185 (vector), pCM-DST1 (DST1) or pCM-ΔN (dst1-133,309). These transformants were transferred on FOA to counterselect the pYX212-RPB4 plasmid, transferred to YPD and incubated for 5 days at 30°C, using ESH29-1B as a control (RPB4). Control experiments showed that pCM-ΔN complements the mycophenolate sensitivity of dst1Δ and is thus functional (data not shown). (C) Synthetic lethality between rpb4D and the dst1-R287Q,E291N mutant defective in the RNA cleavage activity. Strain SL21-3A (rpb4D) was crossed with D495-4D (dst1-R287Q,E291N) and 20 tetrads were analysed. The dst1 rpb4Δ double mutants invariably produced minute colonies that failed to propagate when further restreaked on YPD. One tetratype is shown after 3 days at 30°C.

Model of the interactions relating arrested Pol II, TFIIS, SAGA and the Cdk8 module. (A) Interaction between arrested Pol II, TFIIS and the Cdk8 module. The schematic representation of the Pol II/TFIIS complex is as in Figure 1A. The TFIIS and Med13 interaction recruits the Cdk8 module on an arrested Pol II/TFIIS complex. The Cdk8 kinase may facilitate the TFIIS-dependent cleaving process by temporarily holding back elongation via inhibitory phosphorylation (marked by ‘P') on the CTD. Cdk8 may also phosphorylate TFIIS on its N-terminal domain. (B) Interaction between arrested Pol II, TFIIS and the Spt8-containing form of SAGA. Pol II may become frequently arrested during elongation. Transcript cleavage is then required to resume transcription, in a way that depends on TFIIS and Rpb9. The N-terminal domain of TFIIS (dashed line) binds Spt8 and recruits the Spt8-containing form of SAGA. Based on genetic evidence, this is proposed to be essential in the absence of Rpb4, but does not depend on the Gcn5 histone acetylase. However, Gcn5 may optimise elongation, as suggested by the synthetic lethality between rpb9Δ and gcn5Δ.