Sensitivity of dst1Δ to 6AU and MPA can be partially suppressed by mutations that alter the transcriptional regulation of RP genes. (A) Size distribution of the microcolonies developed by isogenic yeast cells with the indicated genotypes (BY4741 genetic background), in the absence or presence of 25 µg/ml 6AU. Individual cells were microencapsulated in alginate, incubated for 20 h and analysed in a flow cytometer utilizing self-fluorescence as an indicator of cell size, as described in the ‘Materials and Methods’ section. Images of representative microcolonies are shown. Additional microcolonies are shown in Supplementary Figure S3A. (B) Northern blots showing the effect of 6AU (100 µg/ml, 90 min) on the mRNA levels of IMD2 in the indicated isogenic mutants [strains as in (A)]. See quantification in Supplementary Figure S4. Note that a mutant’s ability to suppress the growth defect of dst1Δ in the presence of 6AU does not involve the suppression of the IMD2 up-regulation defect.

The presence of TFIIS in rDNA did not influence the distribution of RNA polymerase I along rDNA. (A) The ChIP experiments reveal a constant binding of TFIIS along rDNA, which partially decreased upon 100 µg/ml 6AU addition. Location of the amplicons utilized for quantitative PCR are shown in Graph. (B) Variation in the distribution of RNA polymerase I along rDNA upon 6AU addition, as measured by the ChIP experiments, using antibodies against a HA-tagged version of the biggest subunit of the enzyme. Rpa190 ChIP data are represented as being normalized to a non-transcribed amplicon within NTS2 (N). Note that addition of 6AU (100 µg/ml) led to a transient decrease in the amount of RNA polymerase I bound to the transcribed region, and that this decrease was equally transient in the absence of TFIIS. All the values represent the average of three independent experiments. Error bars indicate standard deviation. (C) Variation in the specific activity of the RNA polymerases sitting on rDNA caused by 6AU addition (100 µg/ml) to the wild-type and to an isogenic dst1Δ strain. RNA polymerase I specific activity was expressed as the ratio between variation in the transcriptional run-on signal (shown in Supplementary Figure S10A) and variation in the Rpb3 ChIP signal [shown in (B)]. In the latter, amplicons 3 and 5 were used to achieve the ratios in combination with the 18S and 25S run-on signals, respectively.

TFIIS and RNA polymerase II occupancy in response to 6AU. Changes in HA-TFIIS (A) and Rpb3 (B) binding to RNA polymerase II-dependent genes in response to 6AU (100 µg/ml). All the values represent the average of three independent experiments at three different amplicons distributed along the genes. Samples were taken from the same extracts to analyse HA-TFIIS and Rpb3 in parallel. ChIP signals were quantified in relation to the input material. The results of an untranscribed intergenic region (Chromosome V, co-ordinates 9716–9863) are also shown. Error bars indicate standard deviation. (C) Variation of TFIIS/RNA polymerase II ratios upon 6AU (100 µg/ml) addition, as measured by ChIP experiments utilizing antibodies against HA-TFIIS and Rpb3. All the values represent the average of three independent experiments and three different amplicons [shown in (A) and (B)].

TFIIS sustains RNA polymerase II activity in RP genes under transcriptional stress. (A) Variation in the levels of the RNA polymerase II bound to the indicated genes, caused by the addition of 6AU (100 µg/ml) to both the wild-type and an isogenic dst1Δ strain. All the values represent the average of three independent experiments and three different amplicons distributed along the indicated genes (Supplementary Figure S9A and S9B) normalized to time 0. (B) Variation in the specific activity of RNA polymerases sitting on the indicated genes caused by 6AU addition (100 µg/ml) to the wild-type and to an isogenic dst1Δ strain. RNA polymerase II specific activity was expressed as the ratio between the variation in transcriptional run-on signal (Supplementary Figure S10A) and the variation in the Rpb3 ChIP signal [shown in (A)].

TFIIS is required for the balance between rDNA transcripts and RPs mRNAs. Northern blots showing the variation of the 35S primary rRNA precursor and the mRNAs of the indicated genes upon 6AU addition (100 µg/ml) in the wild-type (A) and in the dst1Δ (B), imd2Δ (D), sfp1Δ (E) and dst1Δsfp1Δ (F) isogenic strains. The quantifications for 35S and RPL5 mRNA are shown in order to visualize the 35S/RP mRNA balance. (C) Levels of the 35S, 32S and 27SA rRNA precursors measured by Northern blot upon 6AU addition (100 µg/ml) in the wild-type and in an isogenic dst1Δ strain. Note that the accumulation of 35S detected in dst1Δ is not coupled with a similar accumulation of either 32S or 27SA, as would be expected for a general increase in rDNA transcription. 18S rRNA is shown as the loading control in all the panels.

Deletion of SFP1 almost eliminates the response of RNA polymerase II-dependent genes to 6AU, even in the absence of TFIIS. (A) Variation in the levels of the RNA polymerase II bound to the indicated genes, caused by the addition of 6AU (100 µg/ml) to sfp1Δ and dst1Δsfp1Δ cells. All the values represent the average of three independent experiments and three different amplicons distributed along the indicated genes (Supplementary Figure S9C and S9D). (B) Variation in the specific activity of the RNA polymerases sitting on the indicated genes caused by the addition of 6AU (100 µg/ml) to the sfp1Δ and dst1Δsfp1Δ cells. RNA polymerase II-specific activity was expressed as the ratio between variation in the transcriptional run-on signal (Supplementary Figure S10B) and variation in the Rpb3 ChIP signal [shown in (A)]. Scales were set to facilitate a comparison to Figure 4A and B, respectively.