(A) Growth spotting tests for wild-type (WT) and Kin28 anchor-away (AA) strains on rich YPD media with or without rapamycin at 30 °C for 2 days.
(B) Kin28-FRB occupancy at the indicated promoters in the Kin28-AA and control strains before and after 1 hour of rapamycin treatment. Averages and standard errors of three individual experiments are shown.
(C) Percentage of Pol II occupancy remained at the indicated coding regions in the WT, Spt15-AA and Kin28-AA strains after 1 hour of rapamycin treatment. Averages and standard errors of three individual experiments are presented.
(D) Changes of Pol II occupancy (measured by Pol II ChIP-seq) at 3′ end of active genes in the Kin28-AA and Spt15-AA strains upon rapamycin treatment. Numbers of genes are plotted against the log₂ ratios of Pol II levels after rapamycin treatment (+Rap) over Pol II levels before rapamycin treatment (−Rap).
See also .

(A) Overall Pol II ChIP-seq binding profiles of active genes in WT, Kin28-AA, and Spt15-AA strains before and after 1 hour of rapamycin treatment.
(B) Changes of Pol II processivity (measured between +500–1000) of active genes greater than 1 kb in length in the indicated strains after 1 hour of rapamycin treatment. Changes of Pol II processivity are expressed as log₂ ratios of gradients of the linear regression lines after and before rapamycin treatment.
See also .

(A) TBP and Pol II occupancies at the promoter and the coding regions of three representative genes were analyzed before and after addition of rapamycin for 1 hour in the WT and Kin28-AA strains. Results are expressed as percentage of TBP and Pol II occupancies remained after the rapamycin treatment compared to those without the treatment.
(B) Time course analysis of TBP, Pol II and Kin28-FRB occupancies at the promoter region of highly expressed CCW12 upon rapamycin treatment. Values shown are occupancy levels relative to those before rapamycin treatment (i.e. Time = 0 min). Averages and standard errors of three individual experiments are presented.
See also .

(A, B) Pol II ChIP-seq binding profiles and genome-browser display of the indicated genes in the Kin28-AA strain before (+Kin28) and after (−Kin28) 1 hour of rapamycin treatment. See also .

(A, B) Cumulative frequency plots of transcription (Pol II occupancy) changes at SAGA- and TFIID-dependent genes () upon Kin28 depletion in the Kin28-AA strain or inactivation of Kin28 kinase activity in the kin28-as strain.
(C) Cumulative frequency plots of TFIIB occupancy changes at SAGA- and TFIID-dependent genes upon Kin28 depletion by anchor-away.
(D) Pol II (blue and red lines) and Kin28-FRB (black line) ChIP-seq binding profiles across promoter regions (+/− 500 bp from ATG) of active SAGA- and TFIID-dependent genes in the Kin28-AA strain before and after Kin28 depletion.
(E) Spt16 ChIP-seq binding profiles were plotted for the same regions, as in (D).
See also .

(A) Mediator (Rgr1) occupancy at the core promoters of indicated genes in the Kin28-AA strain before and after Kin28 depletion. Averages and standard errors of three individual experiments are presented.
(B) Comparison between TFIIB levels and Mediator (Rgr1) binding changes in the Kin28-AA strain upon Kin28 depletion. Promoter TFIIB level was determined by the total count of TFIIB ChIP-seq reads within the 100 bp window centered on the transcription start site. For Mediator (Rgr1) binding changes, normalized read counts of analyzed promoters after Kin28 depletion were divided by those before Kin28 depletion and expressed as log₂ ratios. Only promoters with more than 1000 TFIIB raw counts before Kin28 depletion were included in the analysis.
(C) Mediator (Rgr1) and TFIIB ChIP-seq binding profiles of active genes in the Kin28-AA strain before (blue line) and after (red line) Kin28 depletion.
(D) Boxplots of Mediator (Rgr1) to Pol II binding ratios at core promoter regions of active SAGA- and TFIID-dependent genes in the Kin28-AA strain before (+Kin28) and after (−Kin28) Kin28 depletion.
(E) Mediator (Rgr1) occupancy at the core promoters of indicated genes in the kin28-as strain upon inactivation of Kin28 kinase activity by the inhibitor NA-PP1. Averages and standard errors of three individual experiments are presented.
See also .

The general transcription factors and Mediator assemble at a core promoter to form a pre-initiation complex (PIC). Upon PIC formation, Kin28-dependent phosphorylation of Pol II CTD inhibits the interaction between Pol II and Mediator, leading to a rapid dissociation of Mediator from the PIC, facilitating Pol II escape from the PIC for productive elongation. Although it is formally possible that Mediator dissociation and Pol II escape from the PIC occur simultaneously or in the reverse order, we disfavor this possibility as CTD phosphorylation destabilizes the direct Mediator-Pol II interaction in vitro, and there is no evidence in vivo for direct stable Mediator-PIC association in the absence of Pol II. After Pol II promoter escape, a “post-escape” transcription initiation complex, which is relatively stable (minute time scale), remains bound at the core promoter such that transcription re-initiation can occur upon reloading of Mediator and Pol II.