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. 2004 Sep 9;23(21):4232–4242. doi: 10.1038/sj.emboj.7600326

Figure 5.

Figure 5

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.