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. Author manuscript; available in PMC: 2024 Jul 4.
Published in final edited form as: Nat Struct Mol Biol. 2024 Jan 4;31(1):190–202. doi: 10.1038/s41594-023-01171-9

Extended Data Figure 2. Surrounding sequence of TSSs modulates initiation efficiency.

Extended Data Figure 2.

(A) +1 TSS efficiency of all −7 to −2 sequences within each N−8N−1N+1 motif in WT, rank ordered by efficiency of A−8C−1A+1 version shown as a heat map. x-axis is ordered based on median efficiency for each N−8N−1N+1 motif group, as shown in Figure 2B. Spearman’s rank correlation tests between A−8C−1A+1 group and all groups are shown beneath the heat map. (B) Efficiencies of designed +1 TSSs grouped by base identities between −8 and +1 positions. Statistical analyses by Kruskal-Wallis with Dunn’s multiple comparisons test for base preference at individual positions relative to +1 TSS are shown beneath plots. Lines represent median values of subgroups. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (C) Histogram showing the distribution of measured efficiencies for all designed −8 to +4 TSSs of all promoter variants from “AYR”, “BYR” and “ARY” libraries in WT. Dashed lines mark the 5% efficiency cutoff. (D) A+2G+3G+4 motif enrichment is apparent for the top 10% most efficient designed −8 TSS. A(/G)+2G(/C)+3G(/C)+4 motif enrichment was observed for the top 10% most efficient −8 TSSs but not for the next 10% most efficient TSSs. A(/G)+1 enrichment observed for top 20% most efficient TSSs is consistent with the +1R preference of TSS. Numbers (N) of variants assessed are shown. Sequence logos were generated using WebLogo 3. Bars represent an approximate Bayesian 95% confidence interval. (E) An A at position −9 results in different sequence preferences at position −8. The dataset of designed +4 TSSs deriving from “AYR”, “BYR” and “ARY” libraries was used to detect the −9/−8 interaction. All variants were divided into 16 subgroups defined by bases at positions −9 and −8 relative to designed +4 TSS, and then their TSS efficiencies were plotted. Lines represent median values of subgroups. (F) An A at position −8 results in different sequence preferences at position −7. The dataset of designed +1 TSSs deriving from “AYR” and “BYR” libraries was used to detect −8/−7 interaction. Calculations same as −9/−8 interaction described in E.