Figures and data
Tfb3 N-term-Linker fusions to other kinase modules can replace Tfb3, but not Kin28.
(A) Schematic of TFIIH structure within the PIC. The domain structure of Tfb3 (green) is shown, connecting the TFIIH Kinase (cyan) and Core (blue) Modules. (B) Schematic of Tfb3 fusions to other kinase modules. (C) The kin28Δ strain YSB744 was transformed with pRS425 (Vector), pRS415-KIN28, pRS425-TFB3-BUR2, pRS425-TFB3-CTK3, or pRS425-TFB3-MPK1. Transformants were tested for the ability to replace a KIN28/URA3 construct by plasmid shuffling. Cells were streaked on -LEU-TRP (center) or -LEU-TRP+5-FOA (right) plates for 3 days at 30℃. (D) pRS315 (Vector), pRS425-TFB3, pRS315/TFB3Δ2 (aa 1-275) (Feaver et al., 2000), pRS425-TFB3-BUR2, pRS425-TFB3-CTK3, and pRS425-TFB3-MPK1 were transformed into tfb3Δ strain SHY907 (Warfield et al., 2016), replacing the plasmid-expressed wild-type gene by plasmid shuffling. Streaks shown were grown on –LEU for 4 days (center) or on -LEU+5-FOA plates (right) for 9 days.
Tfb3 can be split into two functional parts.
(A) Schematic of Tfb3 deletion constructs. Note that all constructs retained amino acids 1-11 at the N-terminus for ease of cloning and to avoid differential sensitivity to N-end rule degradation. (B) Plasmid shuffling was used to test pRS424 (Vector), pRS424-TFB3 (WT TFB3), pRS424-TFB3(Δ145-321) (N-term), and pRS424-TFB3 (Δ252-321) (N-term-Linker) for the ability to support growth of tfb3Δ shuffling strain SHY907/YF2456 (Warfield et al., 2016). Streaks shown were grown on -TRP+5-FOA plates for 5 days at 30℃ (C) Spot assay for growth. Tfb3 N-term (YSB3704) and N-term-Linker (YSB3722) strains were transformed with pRS425 (Vector), pSH1542 (WT TFB3), pRS315-TFB3(1-11, 238-Stop)-Flag1-TAP (C-term low copy), pRS425-TFB3(1-11, 238-Stop) (C-term high copy), pRS425-TFB3(1-11, 139-Stop) (Linker-C-term low copy), or pLH366 (Linker-C-term high copy). Ten-fold serial dilutions were spotted onto –LEU-TRP plates for 3 days. For comparison to normal growth, an isogenic strain carrying WT TFB3 (YSB3788) was spotted in parallel (top strip).
The TFIIH Kinase Module associates with the untethered Tfb3 C-terminal domain.
(A) Whole cell extracts were made from YSB3788 (TFB3 untagged), YSB3723 (TFB3-TAP + N-term), YSB3732 (N-term-Linker + C-term-TAP), and YSB3728 (N-term + Linker-C-term-TAP). Both Input extracts (lanes 1-4) and IgG-Agarose precipitated fractions (lanes 5-8) were resolved by SDS-PAGE and blotting for Tfb3. (B) To probe for other Kinase Module subunits, IgG precipitates were probed with anti-Ccl1 and anti-Kin28 antibodies. In all panels, asterisks mark TAP-tagged Tfb3 derivatives (which also react with secondary antibody due to the protein A component).
Splitting Tfb3 uncouples the TFIIH Kinase Module from the Core Module and the PIC.
The same IgG-Agarose precipitates analyzed in Fig. 3 were also probed with the following antibodies against: (A) TFIIH Core Module subunit Tfb1 (note background binding of Tfb1 to beads as seen in lane 1, and non-specific band ∼50 kDa), and (B) TFIIE subunits Tfa1 and Tfa2 (note non-specific band ∼60 kDa), which serves as a marker for the RNApII PIC. In all panels, asterisks mark TAP-tagged Tfb3 derivatives reacting with secondary antibody due to the protein A component.
Loss of proper Kinase Module and Ser5P promoter localization in split Tfb3 cells.
Crosslinked chromatin was prepared from strains expressing full length Tfb3 (YSB3788, red line), the separated N-term-Linker and C-term derivatives (YSB3731, yellow), the N-term and Linker-C-term derivatives (YSB3727, blue), or N-term and C-term with no Linker (YSB3725, green). Samples were immunoprecipitated and processed for sequencing of crosslinked DNA as described in Methods. Two biological replicates for each antibody were used to generate metagene profiles of (A) TFIIH Core subunit Tfb1, (B) TFIIH Kinase subunit Kin28, (C) Rpb1 CTD Ser5P, (D) RNApII subunit Rpb1, and (E) Ser5P normalized to total Rpb1. The analysis used only those nuclear genes with an average RNApII occupancy greater than 4 reads per million (rpm) and longer than 1 kb (n=95). Individual replicates are shown in Figure 5 - figure supplement 1. To account for the differing lengths of genes, each graph shows 1 kb centered on the transcription start site (TSS) to the left of the dividing line, and 1 kb centered on the polyadenylation site (pA) to the right.
Split Tfb3 strains are not hyper-sensitive to UV light.
Yeast strains carrying the indicated Tfb3 configurations were plated at various densities and exposed to the indicated dosages of UV light. Surviving colonies were counted after several days. The graph shows the averaged values from multiple replicates, and error bars represent standard deviation. Note that there are two strain backgrounds. The tfb1-101 strain (YSB250, black) is a positive control for an NER-defective strain sensitive to UV light, and its isogenic WT control is YSB207 (yellow). The other five isogenic strains are in a tfb6Δ background (Warfield et al., 2016), lacking the Tfb6 protein that competes with the TFIIH for Ssl2 binding (Murakami et al., 2012): WT/tfb6Δ (YSB3788, green), N-term (YSB3724, red), N-term-Linker (YSB3729, blue), N-term + Linker-C-term (YSB3727, orange), and N-term-Linker + C-term (YSB3731, slate).
Model for linkage between the TFIIH Kinase and Core Module functions.
A. In free TFIIH, the Tfb3 linker contacts the Ssl2/XPB subunit to create a “closed” conformation. In this configuration, the translocase, and possibly the kinase, may be inhibited. B. Upon TFIIH incorporation as the final component of the PIC, the Kinase Module engages with both Mediator (not pictured) and the CTD. These interactions may help release the Tfb3/MAT1 linker from Ssl2/XPB, allowing the translocase to access downstream DNA to promote ATP hydrolysis and promoter melting.
Immunoblot of Tfb3 fusions from Fig. 1C.
Whole extracts were made from the post-FOA strains constructed by shuffling pRS425-TFB3-BUR2, pRS425-TFB3-CTK3, or pRS425-TFB3-MPK1 into tfb3Δ strain SHY907/YF2456 (Warfield et al., 2016). Extracts were resolved by SDS-PAGE, blotted to a membrane, and probed with anti-Tfb3 antiserum. Left-most lane shows markers imaged with visible light, and the rest of the blot shows chemiluminescence detection.
Tethered and untethered Kinase Module have similar CTD kinase specific activity in vitro.
(A) Kinase Module was isolated from YSB3707 (TFB3-TAP), YSB3732 (N-term-Linker + C-term-TAP), and YSB3728 (N-term + Linker-C-term-TAP) using the TAP tag. A parallel isolation from the untagged strain YSB3788 (WT TFB3) showed that binding was dependent on the TAP tag. The second lane shows a Kin28-TAP precipitate (not done in parallel) as size marker. Relative levels of the Kin28 subunit on beads were determined by immunoblotting 8uL, 4uL, and 2uL of beads. (B) In vitro CTD phosphorylation assay. After roughly normalizing bead volumes for amount of bound Kin28, kinase reactions were performed with GST-CTD. Samples were taken at the indicated time points and tested for CTD Ser5 phosphorylation using monoclonal antibody 3E8. Note the phosphorylated CTD as a series of bands, with slower mobility believed to represent denser phosphorylation. To mark the position of unphosphorylated GST-CTD, a negative control reaction lane from the blot was probed with 8WG16 (left panel).
Additional ChIP-seq analysis.
(A-D) These panels are the same metagene analysis as Figure 5A-D, but showing the individual replicates as separate lines. The Tfb3 configurations are indicated in the key at the bottom: wild-type Tfb3 in reds, N-term-Linker + C-term in yellows, N-term + Linker-C-term in blues, and N-term + C-term in greens. (E) For each split Tfb3 mutant (color coded as in A-D), a plot of the mutant/WT log2 RNApII-adjusted Ser5P ratio versus WT RNApII occupancy, for all verified genes (n=5587), over promoters (left) and ORFs (right) (see Methods). In panels E and F, the x-axis is cut at 400 rpm for clarity, excluding only three genes with higher occupancy. (F) Plots of the mutant/WT log2 ratio versus WT level for RNApII occupancy, over ORFs for all verified genes (n=5573). Yellow: N-term-Linker + C-term, Blue: N-term + Linker-C-term, and Green: N-term + C-term. (G) Heatmap of the mutant/WT log2 RNApII ratio for the seven genes that are systematically induced (mutant/WT log2 RNApII ratio>0) in all three split mutants. All but ANS1 are known to be involved in stress-response.
Immunoblot for CTD phosphorylation levels in strains with split Tfb3 or completely lacking the C-term.
Whole cell extracts from YSB3712 (WT), YSB3710 (N-term), YSB 3711 (N-term-Linker), YSB3731 (N-term-Linker + C-term), and YSB3727 (N-term + Linker-C-term) were resolved by SDS-PAGE and immunoblotted. The top blot shows CTD Ser5P (monoclonal antibody 3E8), the middle blot shows Ser2P (monoclonal antibody 3E10), and the bottom blot shows both Rpb1 (monoclonal antibody 8WG16) and Rpb3 (monoclonal antibody 1Y26).
Individual gene traces from the ChIP-seq analysis show Ser5P changes in split Tfb3 strains.
A. Rpb1 and Ser5P signals at a region containing five strong promoters (shaded in green) were plotted using the UCSC browser (https://genome.ucsc.edu). Blue bars above traces show gene locations, with arrows showing direction of transcription. Numbers in the right-hand corner show the read count scale of the y-axis. B. Traces of total Rpb1, Ser5P, and Ser2P at the strongly transcribed PMA1 gene. Annotations are as in part A.
Effects of Tfb3 split mutants on RNApII occupancy are consistent across different gene classes.
A. Metagene analysis of RNApII occupancy at “long” genes, defined as genes longer than 2 kb and having an average RNApII occupancy greater than 2 reads per million (rpm) (n=41). B. Metagene analysis of RNApII occupancy over all genes longer than 1 kb separated in quintiles, Q1 representing genes with the highest RNApII occupancy and Q5 the lowest. C. Metagene analysis of RNApII occupancy over coactivator redundant (CR) genes (top; n=151) and TFIID-regulated genes (bottom; n=55) longer then 1 kb. Gene classifications are from Donczew et al. (2020). The Tfb3 configurations are indicated in the key at the bottom: wild-type Tfb3 in red, N-term-Linker + C-term in yellow, N-term + Linker-C-term in blue, and N-term + C-term in green. To account for the differing lengths of genes, each graph shows 1 kb centered on the transcription start site (TSS) to the left of the dividing line, and 1 kb centered on the polyadenylation site (pA) to the right.
