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. 2021 Oct 15;10:e71013. doi: 10.7554/eLife.71013

Figure 6. Genetic interactions between ssl2 and polymerase II (Pol II) initiation alleles suggest distinct functions of each in initiation by scanning.

(A) Growth phenotypes of rpb1, ssl2 N230D, ssl2 N230I single or double mutants. rpb1 mutants represent known catalytically hyperactive alleles or genetically similar (G1097D, E1103G, L1101S, F1084I) and four with reduced catalytic activity (F1086S, H1085Q, N1082S, H1085Y). Strains are arranged according to measured Pol II elongation rate in vitro (slowest at top). (B) ssl2 mycophenolic acid (MPA)-sensitive alleles are epistatic to Pol II LOF alleles’ His+ phenotypes (double mutants retain MPAS of ssl2 single mutant while His+ phenotypes of rpb1 mutants are suppressed). Conversely, Pol II transcription start site (TSS) upstream shifting alleles appear epistatic/non-additive with ssl2 MPAS alleles and do not show synthetic growth phenotypes. (C) Pol II upstream TSS shifting alleles appear epistatic to ssl2 N230I phenotypes (MPAS retained and His+ suppressed in double mutants). There are only minor synthetic defects between ssl2 N230I and Pol II downstream TSS shifting mutants suggesting lack of synergistic defect and either mild additivity or epistasis. (Double mutant of N230I and H108Y is nearly dead and was not tested here or in E.) (D,E) Schematic (D) indicating how qualitative growth data of mutants encoded (E) for visualization in heatmaps. (F) Phenotyping heatmap legend. (G) Qualitative heatmaps for ssl2 and rpb1 genetic interactions. Growth phenotypes are detected using reporters described in Figure 1. (H) Primer extension of ssl2 N230D and rpb1 mutants at ADH1. ssl2 N230D appears to truncate distribution of TSSs on downstream side and is epistatic to downstream shifting rpb1 alleles (blue bar) while upstream shifting rpb1 alleles (green bar) are non-additive or epistatic to ssl2 N230D. Numbered regions indicate TSS positions that were binned for quantification in (I). Representative primer extension of ≥3 independent biological replicates is shown. (I) Quantification of (H) with heatmap showing relative differences in TSS distribution binned by position (bins are numbered and shown in H). Mean changes of ≥3 independent biological replicates are shown in the heatmap. (J) Primer extension of ssl2 N230I and rpb1 mutants at ADH1. ssl2 N230I appears to enhance usage of downstream TSSs and is additive with downstream shifting rpb1 alleles (blue bar) while upstream shifting rpb1 alleles (green bar) are epistatic to ssl2 N230I. Numbered regions indicate TSS positions that are binned for quantification in (K). Representative primer extension of ≥3 independent biological replicates is shown. (K) Quantification of (J) with heatmap showing relative differences in TSS distribution binned by position (bins are numbered and shown in (J)). Mean changes of ≥3 independent biological replicates are shown in the heatmap.

Figure 6—source data 1. Figure 6G Heatmap data.
Figure 6—source data 2. Figure 6H Primer extension gel (annotated).
Figure 6—source data 3. Figure 6H Primer extension gel (annotated).
Figure 6—source data 4. Figure 6I rpb1 single heatmap data.
Figure 6—source data 5. Figure 6I N230D single heatmap data.
Figure 6—source data 6. Figure 6I N230D double heatmap data.
Figure 6—source data 7. Figure 6J Primer extension gel (annotated).
Figure 6—source data 8. Figure 6J Primer extension gel (annotated).
Figure 6—source data 9. Figure 6K rpb1 single heatmap data.
Figure 6—source data 10. Figure 6K N230I single heatmap data.
Figure 6—source data 11. Figure 6K N230I double heatmap data.
Figure 6—source data 12. Figure 6H Primer extension gel (raw).
Figure 6—source data 13. Figure 6H Primer extension gel (raw).
Figure 6—source data 14. Figure 6J Primer extension gel (raw).
Figure 6—source data 15. Figure 6J Primer extension gel (raw).

Figure 6.

Figure 6—figure supplement 1. Design of ssl2 genetic interaction tests with efficiency alleles.

Figure 6—figure supplement 1.

(A) Schematic showing a promoter window with transcription start sites (TSSs). (B) Increased processivity is hypothesized to increase TSS usage downstream. (C) Decreased processivity is hypothesized to reduce TSS usage downstream. (D) Increased efficiency is expected to activate TSS usage upstream. (E) Decreased efficiency is expected to increase apparent TSS usage downstream. (F) A processivity gain-of-function (GOF) allele and efficiency GOF double mutant is expected to show efficiency GOF single mutant allele’s effects on activating upstream TSSs (epistasis). (G) A processivity GOF and efficiency loss-of-function (LOF) double mutant is expected to allow or enhance efficiency LOF single allele’s effect on increasing apparent TSS usage downstream (potential additivity). (H) A processivity LOF and efficiency GOF double mutant is expected to show efficiency GOF single allele’s effect on activation TSS usage upstream (epistasis). (I) A processivity LOF and efficiency LOF double mutant is expected to show processivity LOF single allele’s effect on reducing downstream TSS usage (epistasis).
Figure 6—figure supplement 2. The scoring method used to quantify yeast growth phenotypes and make phenotypic heatmaps.

Figure 6—figure supplement 2.

Figure 6—figure supplement 3. Polymerase II (Pol II) efficiency alleles are able to increase transcription start site (TSS) efficiency within the processivity defined scanning window.

Figure 6—figure supplement 3.

(A) Quantification of TSS usage at ADH1 in rpb1 single mutant, ssl2 N230D single mutant and the double mutants. TSS usage in single or double mutants is compared to wild-type (WT) TSS usage. Bars are average ± standard deviation of ≥3 independent biological replicates. (B) Quantification of TSS usage at ADH1 in rpb1 single mutant, ssl2 N230I single mutant and the double mutants. TSS usage in double mutants is compared to WT TSS usage. Bars are averages ± standard deviation of ≥3 independent biological replicates.
Figure 6—figure supplement 3—source data 1. Figure 6—figure supplement 3A Graph data.
Figure 6—figure supplement 3—source data 2. Figure 6—figure supplement 3B Graph data.