Evidence for a complex of transcription factor IIB with poly(A) polymerase and cleavage factor 1 subunits required for gene looping

Abstract

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

FIGURE 1.

CF1 subunits and poly(A) polymerase copurify with TFIIB on an anti-HA affinity column. A, HA-tagged TFIIB was affinity-purified from cells harboring Myc-tagged CF1 subunits or poly(A) polymerase as described under “Experimental Procedures.” Purified samples were subjected to SDS-PAGE followed by Western blot analysis using anti-HA and anti-Myc antibodies. Lane 1 displays molecular weight (MW) marker proteins, and lane 2 represents Imperial Coomassie Blue staining of the eluate from an anti-HA affinity column. B, affinity purifications were performed for HA-tagged TFIIB in the wild type and the looping-defective mutant sua7-1 strain. The eluates from the affinity columns were subjected to SDS-PAGE, and Western blotting was performed with antibodies against TFIIB, Rna15, and poly(A) polymerase. C, affinity-purified HA-tagged TFIIB was subjected to SDS-PAGE followed by Western blot analysis using anti-HA, anti-TBP, anti-Kin28, and anti-Rpb1 antibodies. IP, immunoprecipitation.

FIGURE 2.

Sedimentation analysis of TFIIB. A, sedimentation analysis reveals copurification of CF1 subunits and poly(A) polymerase with TFIIB. Affinity-purified HA-tagged TFIIB was subjected to sedimentation analysis in 5–30% (v/v) glycerol gradient in 150 mm KCl. Fractions of 1.8 ml each were collected and subjected to SDS-PAGE analysis followed by Western blotting to visualize TFIIB, CF1 subunits, and poly(A) polymerase. Input bands indicate affinity-purified sample prior to sedimentation analysis. B, sedimentation analysis of affinity-purified TFIIB from wild type cells, rTFIIB, and affinity-purified TFIIB from looping-defective sua7-1 cells under the conditions as described above.

FIGURE 3.

Holo-TFIIB complex is susceptible to high ionic strength and sediments between TFIID and TFIIH complexes. A, affinity-purified HA-tagged TFIIB and rTFIIB were subjected to sedimentation analysis in 5–30% (v/v) glycerol gradient in 500 mm KCl. Fractions were collected and processed as in Fig. 2. B, affinity-purified preparations of TFIIB (panel i), TBP (panel ii), and Kin28 (panel iii) were subjected to sedimentation analysis in 5–30% (v/v) glycerol gradient in 150 mm KCl as described previously. Fractions were collected, processed as above, and probed for TFIIB and TBP or TFIIB and Kin28.

FIGURE 4.

Gene looping requires the CF1 complex and poly(A) polymerase. A, schematic representation of MET16 and INO1 indicating the positions of AluI restriction sites (vertical lines) and PCR primers (arrows) used in CCC analysis. B, CCC analysis of MET16 and INO1 to detect gene looping in W303-1a (wild type) and mutant strains of Rna14 (rna14-1), Pcf11 (pcf11-2), Hrp1 (hrp1-5), and poly(A) polymerase (pap1-1) following 120 min of induction followed by incubation at either permissive (25 °C) or non-permissive (37 °C) temperatures. P1T1 PCR reflects the looping signal, whereas Control PCR represents the loading control indicating that equal amount of template DNA was present in each of the CCC reactions. C, quantification of the CCC results shown in B, representing ChIP signal/Input signal. Error bars indicate one standard deviation.

FIGURE 5.

TFIIB cross-linking to the terminator region is dependent upon a functional CF1 complex and poly(A) polymerase. A, schematic depiction of INO1 indicating the positions of ChIP primer pairs A, B, C, and D. B, ChIP analysis showing cross-linking of TFIIB to different regions of INO1 in W303-1a (wild type) and mutant strains of Rna14 (rna14-1), Pcf11 (pcf11-2), Hrp1 (hrp1-5), and poly(A) polymerase (pap1-1) following 120 min of induction followed by incubation at either permissive (25 °C) or non-permissive (37 °C) temperatures. The Input signal represents DNA prior to immunoprecipitation. C, quantification of the data shown in B, representing ChIP signal/input signal. Error bars indicate one standard deviation.

FIGURE 6.

The CF1 complex and poly(A) polymerase cross-link to the promoter and the terminator regions of MET16 and INO1 during activated transcription. A and B, schematic depiction of MET16 and INO1 indicating the positions of ChIP primer pairs A, B, C, and D. C and D, quantification of the ChIP analysis data showing cross-linking of Rna14, Pcf11, Hrp1, and Pap1 to different regions of MET16 and INO1 during repressed (black bars) and activated (gray bars) transcription. Met, methionine; Ino, inositol. Error bars indicate one standard deviation.

FIGURE 7.

Induced transcription in the looping-defective mutant of TFIIB exhibits a kinetic lag. A and D, schematic depiction of MET16 and INO1 indicating the positions of RT-PCR primer pairs. B and E, RT-PCR analysis of MET16 and INO1 in WT and looping-defective sua7-1 strains following transfer of cells to transcription-inducing conditions at the indicated time points. C and F, quantification of the data shown in B and E. Error bars indicate one standard deviation.