Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II

Abstract

As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal transcription factors TFIID, TFIIA, TFIIH, and TFIIE remain at the promoter as part of a scaffold complex, whereas TFIIB and TFIIF dissociate. The yeast Ctk1 kinase associates with elongation complexes and phosphorylates serine 2 in the YSPTSPS repeats of the Rpb1 C-terminal domain, a modification that couples transcription to mRNA 3'-end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5'-end of genes in the transition from initiation to elongation. In strains lacking Ctk1, many basal transcription factors cross-link throughout transcribed regions, apparently remaining associated with RNApII until it terminates. Consistent with this observation, preinitiation complexes formed on immobilized templates with transcription extracts lacking Ctk1 leave lower levels of the scaffold complex behind after escape. Taken together, these results suggest that Ctk1 is necessary for the release of RNApII from basal transcription factors. Interestingly, this function of Ctk1 is independent of its kinase activity, suggesting a structural function of the protein.

Figure 1

Ctk1 is necessary for the dissociation of basal transcription factors from elongating RNA polymerase II (RNApII). (A) Schematic of the PMA1, ADH1, and PYK1 genes. The UAS of each gene is indicated by an open box (see Figure 3C). The TATA/promoter region and open reading frames are represented by black and grey boxes, respectively. Arrows indicate the position of the major polyadenylation sites reported previously (Kim et al, 2004a) and bars below the genes show the relative positions of PCR products in ChIP analysis. (B) Occupancy of Rpb1 and basal transcription factors (Sua7, TBP, Kin28, Tfb1, Tfa2) at the indicated regions in WT (YSB726) or ctk1Δ (YSB854) cells. INPUT was used to normalize the PCR amplification and the asterisk marks a non-transcribed PCR fragment indicated in all reactions as a background control. (C) Quantitation of the ChIP experiments in (B), with PMA1 as representative. The x axis indicates the specific primer pair used in each PCR. The y axis shows the specific signal relative to the negative control (i.e., a ratio of one is equivalent to background).

Figure 2

The abnormal cross-linking of basal transcription factors in ctk1Δ cells is confirmed by independent immunoprecipitation of TAP-tagged TFIIF subunits. (A) ChIP analysis was carried out using Rpb3–TAP (YSB956) or Tfg1–TAP tagged strains (YSB925) +/− Ctk1. Similar cross-linking patterns were seen with Tfg2–TAP (YSB926) or Tfg3–TAP (YSB927) strains (not shown). IgG agarose was used for immunoprecipitation of TAP-tagged proteins. All primer pairs used are described in Figure 1A. PCR products are shown as in Figure 1B. (B) Quantitation of results in (A).

Figure 3

Basal transcription factors coincide with elongating RNApII in ctk1Δ cells. (A) ChIP analyses were carried out with antibodies against Rpb3, TBP, or Kin28 in WT (YSB726) and ctk1Δ (YSB854) backgrounds. Numbers (6–9) correspond to PMA1 primer locations in Figure 1A. PCR products from (A) and (C) are shown in Figure 1B. (B) Quantitation of results in (A). (C) Occupancies of Rap1, Rpb1, and TBP at the indicated regions of genes were determined using the indicated polyclonal antibodies in both WT (YSB726) and ctk1Δ (YSB854) backgrounds. The upstream activating sequence (UAS) regions of each gene are depicted in Figure 1A.

Figure 4

Ctk1 kinase activity is not required for the dissociation of basal transcription factors. (A) Three other cyclin-dependent kinases involved in transcription do not regulate the 5′ transitions. Cells with kin28-T17D (YSB592) or srb10-D290A (YF243) alleles were grown at 30°C and prepared for ChIP analysis. YSB524 is isogenic with YSB592 but contains a WT KIN28-covering plasmid and was used as a WT control. The bur1-23 strain (YSB1021) was grown at 25°C and shifted to 37°C (non-permissive temperature) for 4 h to severely reduce its kinase activity. Chromatin from each strain was immunoprecipitated with anti-TBP. PCR products from (A), (B), (D), and (E) are shown as in Figure 1B. (B) Serine 2 phosphorylation of the Rpb1 CTD does not regulate the 5′ transitions. ChIP was carried out in the indicated strains. pRP112 (RPB1 WT plasmid) in YF68 was replaced by pRP114 (RPB1), pY1WT(10) (rpb1 with 10 WT repeats) or pY1A²(8)WT(7) (rpb1 with seven WT and eight S2A repeats). Each strain was then transformed with RNA14–TAP (creating YSB1188, YSB1189, and YSB1190, respectively) before ChIP analysis. Protein A- or protein G-sepharose was used for TBP or Rpb3 immunoprecipitation and rabbit IgG agarose was used for RNA14–TAP pull-down. (C) Quantitation of results from (B). (D) A strain with an rpb1-1 temperature-sensitive allele (YSB784) was transformed with pRP114 (RPB1 WT copy) or pY1A²(14) (Rpb1 with 14 S2A mutant repeats of CTD). Cells were grown at 22°C, shifted to 37°C (non-permissive temperature) for 4 h as indicated, and prepared for ChIP analysis. (E) The Ctk1-deficient strain (YSB854) was transformed with either of the plasmids YCplac22 (empty vector), YCplac22-CTK1HA (T338A), YCplac22-CTK1-DN, or pRS316-CTK1 and used for ChIP analysis.

Figure 5

Ctk1 is required for the stability of the scaffold. (A) Immobilized templates used in this study. The HIS4 template contains a single Gal4 DNA-binding site upstream of the HIS4 core promoter containing a TATA box and transcription start site. The Promoterless template retains the Gal4 DNA-binding site but lacks the HIS4 promoter. Templates were immobilized on magnetic Dynabeads. (B) The scaffold formation assay. Cell extracts were incubated with the immobilized templates and activator Gal4-VP16 for 40 min. For single round measurement of transcription, PICs were incubated with NTPs for 2 min. (C) Immobilized templates were incubated with the activator Gal4-VP16 and yeast whole cell extracts from either WT (YSB726) or ctk1Δ (YSB854) for 40 min. After the formation of PICs, either nucleotides or ATP was added and incubated for 2 min. The templates were then washed and isolated by digestion with PstI and detected by western blot. Lanes 1 and 5 show the controls for non-specific binding without promoter. Lanes 2 and 6 show typical PIC formation at the promoter. (D) Efficiency of scaffold formation was measured by quantitation of band intensities from lane 3 or 7 in WT and ctk1Δ cells (ImageJ v1.32). Signals from lane 2 or 6 were used as a control for each quantitation. Error bars are from three independent repetitions. (E) The scaffold assay after transcription termination. The cell extracts from either WT (YSB726) or ctk1Δ (YSB854) were incubated with the HIS4-immobilized templates and Gal4-VP16 for 40 min. To measure the association between RNApII and the basal transcription factors after termination, the supernatant was removed after incubation with NTPs for 2 min and immunoprecipitated with anti-Rpb3 antibody. (F) Coimmunoprecipitated proteins with Rpb3 from WT and ctk1Δ cells were determined by western blot using antibodies against non-scaffold (Tfg2 or Sua7) and scaffold components (TBP, Tfb1, Kin28, or Tfa2). (G) Model for the dissociation of basal transcription factors from elongating RNApII. See discussion for details.