CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3'-end processing factor, Pcf11 - PubMed (original) (raw)

CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3'-end processing factor, Pcf11

Zhiqiang Zhang et al. Genes Dev. 2005.

Abstract

Pcf11 is one of numerous proteins involved in pre-mRNA 3'-end processing and transcription termination. Using elongation complexes (ECs) formed from purified yeast RNA polymerase II (Pol II), we show that a 140-amino acid polypeptide from yeast Pcf11 is capable of dismantling the EC in vitro. This action depends on the C-terminal domain (CTD) of the largest subunit of Pol II and the CTD-interaction domain (CID) of Pcf11. Our experiments reveal a novel termination mechanism whereby Pcf11 bridges the CTD to the nascent transcript and causes dissociation of both Pol II and the nascent transcript from the DNA in the absence of nucleotide hydrolysis. We posit that conformational changes in the CTD are transduced through Pcf11 to the nascent transcript to cause termination.

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Figures

Figure 1.

Figure 1.

Generation of a stalled EC from purified yeast Pol II and a tailed template. (A) Schematic of the stalled EC formed from highly purified yeast Pol II and a special template. In the presence of the UpG dinucleotide, purified Pol II initiates transcription at the underlined AC and elongates to the end of a 68-nt-long G-less cassette (for further description, see Zhang et al. 2004). The bottom strand has a biotin tag at its 5′-end for immobilizing the DNA template. (B) Evaluation of transcripts produced under various conditions. All reactions contained 500 μM ATP, 500 μM UTP, and 25 μM UTP supplemented with 2 μCi of α-labeled 32P UTP. For samples in lanes 2 and 3, RNase treatments were performed on stalled ECs. (C) The stalled ECs resume elongation when supplied GTP. (Lane 1) Transcripts recovered from complexes stalled by the absence of GTP. The weak band marked by an asterisk appears to be due to contaminating GTP, since it can be eliminated by including 3′-methoxy-GTP in the reaction (Zhang et al. 2004). (Lane 2) Run-off transcripts produced when stalled ECs were supplied GTP. Radioactive transcripts were detected in denaturing gels with a PhosphorImager.

Figure 2.

Figure 2.

The CID of Pcf11 dismantles an EC. (A) Native gel analysis of ECs treated with intact Pcf11, the CID, or mutant derivatives. The amino acid changes associated with the M1, M2, and M3 mutations are provided above lanes 4_–_6. (Lane 1) ECs generated from purified Pol II and treated with BSA. (Lanes 2_–_6) ECs treated for 30 min with Pcf11, CID, or its derivatives prior to electrophoresis on the native gel. The complexes were detected by the presence of the radiolabeled nascent transcript. (B) Measurement of transcripts released from immobilized ECs following treatment with derivatives of Pcf11. The ECs were formed on DNA templates that had been immobilized on magnetic beads via an avidin-biotin linkage (Zhang et al. 2004). The immobilized ECs were incubated for 30 min with various derivatives of Pcf11 and then separated into bound (B) and released (R) fractions. The radiolabeled transcripts in each fraction were isolated and analyzed on a denaturing polyacrylamide gel. (C) Western blot analysis of the Pol II recovered from the bound and released fractions following treatment of immobilized ECs with derivatives of the CID. Pol II was detected in the bound and released fractions using the Pol II antibody, ARNA-3 (Research Diagnostics, Inc.).

Figure 3.

Figure 3.

The effects of the CID on Pol IIA and Pol IIB ECs. (A) Detection of Pol IIA and Pol IIB ECs before and after treatment with CID. Prior to forming ECs, a portion of Pol IIA was converted to Pol IIB by treatment with chymotrypsin. Another portion of the Pol IIA was treated first with PMSF and then chymotrypsin, so no proteolysis occurred. (Lane 1) Pol IIA EC treated with buffer. (Lanes 2,3) Pol IIA EC formed from Pol IIA that had been incubated first with PMSF followed by chymotrypsin. The EC in lane 3 received additional treatment with the CID prior to electrophoresis. (Lanes 4,5) Pol IIB EC formed from Pol IIB generated by treatment first with chymotrypsin followed by PMSF. The EC in lane 5 received additional treatment with CID prior to electrophoresis. (B) Western blot analysis detecting the shift in mobility of the largest Pol II subunit following removal of the CTD.

Figure 4.

Figure 4.

Evidence that Pcf11 dismantles the EC by bridging the CTD to the nascent transcript. (A) Coomassie blue-stained gel showing purified preparations of derivatives of the CID. (B) Equal amounts of the CID (lanes 1,2) and CID-M2 derivative (lanes 3,4) were incubated with immobilized GST-CTD (lanes 1,3) or GST (lanes 2,4). After washing to remove unbound protein, the bound material was analyzed by SDS-PAGE and Western blotting with antibody against the 6-histidine tag associated with CID and its derivative. (C) CTD pull-down analysis for binding between CID derivatives and the CTD. GST-CTD fusion protein with a 6-histidine tag at the C terminus was immobilized on glutathione Sepharose and separated into four aliquots. Each aliquot of immobilized CTD was incubated with a different derivative of the CID. The immobilized material was extensively washed and then analyzed by Western blotting with antibody against the 6-histidine tag residing on both the CID and GST-CTD. (D) Protein–RNA cross-linking assay for CID–RNA binding. One microgram of each CID derivative was incubated with radiolabeled RNA and cross-linked with UV light for 2 min. The RNA was degraded with RNase A and proteins retaining small radioactive tags were detected after SDS-PAGE using a PhosphorImager. (E) CID forms a bridge between RNA and an immobilized form of the CTD. Glutathione Sepharose beads were first loaded with GST-CTD (lanes 1_–_5), or GST-CTD (lane 6). Following several washes, the beads were incubated with CID and its derivatives as indicated above the lanes. Beads were again washed and then incubated with radiolabeled RNA followed by washing to remove unbound RNA. The radiolabeled RNA was isolated from the beads and analyzed on a denaturing polyacrylamide gel. Lane 7 shows 10% of the total amount of RNA added to each of the samples.

Figure 5.

Figure 5.

DNA oligonucleotide blocking test of a Pcf11-dependent dismantling mechanism. (A) A possible mechanism by which Pcf11 disrupts the EC. By forming a bridge between the CTD and the nascent transcript, movement of the CTD might be transduced via the Pcf11 to the nascent transcript, and the resulting force on the nascent transcript could disrupt the EC. See text for further discussion. (B) Illustration of the DNA oligonucleotide blocking experiment. DNA oligonucleotides (Oligo) 1, 2, and 3 are complementary to parts of the nascent transcript corresponding to regions 1, 2, and 3, respectively (sequences are provided in Materials and Methods). (C) ECs remaining after pretreatment with DNA oligonucleotides followed by CID treatment. ECs were formed and then incubated for 10 min with buffer (lane 1), all three DNA oligonucleotides (lane 2), or one of the three individual oligonucleotides (lanes 3_–_5). Next, buffer (lanes 1,2) or 3 μg of CID was added to the EC, and the mixtures were incubated an additional 30 min before analyzing the ECs on a native gel.

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