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

Figure 9. Model for interaction between initiation efficiency and scanning processivity.

(A) The ‘Shooting Gallery’ model. The polymerase II (Pol II) active site controls initiation efficiency, that is, ‘the rate of firing’. TFIIH controls the rate and extent of scanning, that is, ‘the speed of target passage and number of targets reached’. (B) Reduction in relative transcription start site (TSS) usage as scanning Pol II initiates. As Pol II (wild-type [WT]) scans from upstream to downstream, successful initiation at upstream positions will reduce the amount of Pol II continuing to scan downstream. Increasing initiation efficiency at each position as is predicted for increased Pol II catalytic activity will result in a more rapid decrease in observed initiation from upstream to downstream. Conversely, reducing initiation efficiency at each position will flatten observed TSS distribution because more Pol II will reach downstream positions. (C) TSS distributions during promoter scanning in the ‘Shooting Gallery’ model. The TSS distribution (black arrows) of a promoter window can be affected by Pol II catalytic activity, preinitiation complex (PIC) scanning rate and processivity, TSS strength, Pol II flux, and additional observed (upstream limitation on initiation too close to PIC assembly) or potential (downstream limitation through chromatin structure) constraints. (D) Effects of Pol II catalytic activity on TSS distributions. Increased Pol II catalytic activity increases the efficiency of upstream TSSs that are encountered by Pol II and decreases the usage of downstream TSSs due to quickly reduced Pol II flux (changes indicated as green arrows). Decreased Pol II catalytic activity decreases TSS efficiency of upstream TSSs encountered by Pol II and increases apparent TSS usage at downstream sites due to failed upstream initiation, resulting in a downstream shifted TSS distribution within a window determined by PIC scanning potential (changes shown as blue arrows). (E) Effects of altered scanning processivity on TSS distributions. Increased processivity alleles are hypothesized to increase the probability of Pol II scanning further downstream if Pol II flux remains, thus expanding the scanning window and allowing Pol II usage of downstream TSSs if Pol II flux is not limiting (orange TSS). In contrast, decreased processivity will limit Pol II scanning downstream, truncating the distribution of observed TSSs (purple TSS).

Figure 9.

Figure 9—figure supplement 1. ssl2 alleles shift preinitiation complex (PIC)-component positioning genome-wide as predicted for mutants altering scanning processivity.

Figure 9—figure supplement 1.

(A) Positions of PIC components Sua7 and Ssl2 determined by ChIP-exo in wild-type (WT) and ssl2 mutant strains. Aggregated genome-wide ChIP-exo signals on top (TOP) or bottom (BOT) strand for transcription start site (TSS) ± 150 nt centered promoter windows. Enrichment of ChIP-exo signals from the top or bottom strand of Sua7 or Ssl2 sites are shown for two biological replicates per genotype. Curves are LOWESS smoothing of aggregated ChIP-exo reads from the top 50% promoters in WT. (B) ssl2 alleles shift positioning of PIC components Sua7 and Ssl2 at most promoters. Shown is the shift of median Sua7 or Ssl2 position determined by ChIP-exo as illustrated in (A). Statistical analysis shows that the shift of the average median positions of Sua7 and Ssl2 in the two replicates is significantly different from zero (Wilcoxon signed rank test) at ****p < 0.0001.
Figure 9—figure supplement 1—source data 1. Figure 9—figure supplement 1A Graph data.
Figure 9—figure supplement 1—source data 2. Figure 9—figure supplement 1B Graph data.
Figure 9—figure supplement 2. Effects of TAP-tagging SSL2.

Figure 9—figure supplement 2.

A TAP-tag was integrated at the 3’ end of SSL2 and CRISPR-Cas9 was used to introduce mutations at codon 230 along with an adjacent silent PAM site mutation to prevent Cas9 recutting. It was discovered that the TAP-tag modulated SSL2 phenotypes, behaving like a slight loss of function, causing mild mycophenolic acid (MPA) sensitivity in WT, enhancing MPA sensitivity of ssl2 N230D (top row, SC + MPA plate), suppressing the Spt- phenotype of N230I (SC-Lys plates), but having no effect on the His+ phenotype of N230I in the background of the imd2∆::HIS3 initiation reporter (bottom row, SC-His, SC-His 1 mM 3-AT). 3-Aminotriazole (3-AT) is a competitive inhibitor of the HIS3 gene product. All serial dilution phenotyping results are representative of at least two independently constructed strains (biological replicates).