Dismantling promoter-driven RNA polymerase II transcription complexes in vitro by the termination factor Rat1

Abstract

Proper RNA polymerase II (Pol II) transcription termination is essential to generate stable transcripts, to prevent interference at downstream loci, and to recycle Pol II back to the promoter (1-3). As such, termination is an intricately controlled process that is tightly regulated by a variety of different cis- and trans-acting factors (4, 5). Although many eukaryotic termination factors have been identified to date, the details of the precise molecular mechanisms governing termination remain to be elucidated. We devised an in vitro transcription system to study specific Pol II termination. We show for the first time that the exonucleolytic Rat1·Rai1 complex can elicit the release of stalled Pol II in vitro and can do so in the absence of other factors. We also find that Rtt103, which interacts with the Pol II C-terminal domain (CTD) and with Rat1, can rescue termination activity of an exonucleolytically deficient Rat1 mutant. In light of our findings, we posit a model whereby functional nucleolytic activity is not the feature of Rat1 that ultimately promotes termination. Degradation of the nascent transcript allows Rat1 to pursue Pol II in a guided fashion and arrive at the site of RNA exit from Pol II. Upon this arrival, however, it is perhaps the specific and direct contact between Rat1 and Pol II that transmits the signal to terminate transcription.

Keywords: Enzyme Mechanisms; Molecular Biology; RNA Polymerase II; Rat1; Transcription Termination; Yeast Transcription.

FIGURE 1.

Characterization of stalled Pol II complexes. A, diagram of template 1. The G-less templates are amplified with a pair of primers, one of which is biotinylated (checkered circle). Template 1 consists of a 490-bp upstream region, including the GAL4 upstream activation sequence and the CYC1 TATA element, followed by the 65-bp G-less cassette and a downstream 460-bp G-rich sequence. The template is bound to streptavidin-coated magnetic beads (hatched-filled circle) for assembly of pre-initiation complexes, followed by transcription in the absence of GTP. B, analysis of stalled and extended transcription products. GTP-starved transcripts synthesized from template 1 are body-labeled with [α-P³²]UTP (-GTP) and separated on a 7 m urea/8% polyacrylamide gel. The major transcription product (65 nt) is indicated with an arrow. The minor transcription product (80 nt), indicated with an asterisk, is the result of an alternative start-site selection. Stalled transcripts are chased with the addition of 1 mm of all NTPs (+ GTP). As a control, pre-initiation complexes are incubated with 10 μg/ml α-amanitin prior to the addition of all four NTPs. The sizes of radioactive RNA markers (lane M) are given in nucleotides on the left. C, Western blot of Pol II molecules in pre-initiation (Int) and post-transcription (Txn) complexes, as diagrammed on the right. Detection is with anti-hypo-P Rpb1 CTD (8WG16) (left panel) and anti-Ser⁵-P Rpb1 CTD (H14) (right panel).

FIGURE 2.

Nascent complex-bound transcripts are sensitive to Rat1·Rai1 in a salt-dependent manner. A, purified recombinant wt Rat1·Rai1 resolved on a 12% SDS-polyacrylamide gel stained with silver. B, effect of buffer on Rat1 exonuclease activity. Complexes (∼ 2 fmol) stalled on template 1 (Fig. 1A) were challenged with 75 nm Rat1·Rai1 complex in transcription buffer (glutamate buffer, left panel) or with exonuclease buffer (chloride buffer, right panel). The transcript is examined for its appearance in the bound (B) or in the released (R) fractions upon magnetic precipitation of the DNA template. The amount of RNA remaining in the bound complexes is given as a percentage of that from the mock reactions lacking Rat1·Rai1. C, Western blot analysis of buffer effects on Rat1-mediated Pol II release. Pol II complexes present on template 1 after transcription in the absence of GTP were challenged with 75 nm Rat1·Rai1 complex in the indicated buffers. The bound (B) and released (R) fractions were resolved on an 8% SDS-polyacrylamide gel, transferred to a PVDF membrane, and blotted with the anti-pan-CTD antibody 4H8. D, Western blot of Pol II pre-initiation (left four lanes) and post-transcription (right four lanes) complexes challenged with Rat1·Rai1. Pol II is examined as described in C. The percent of released Pol II after Rat1·Rai1 treatment is indicated.

FIGURE 3.

Mapping the distribution of Pol II complexes along the DNA template. A, diagram of template 2. Template 2 incorporates 40 bp derived from sequence around the CYC1 poly(A) site (Ext) immediately upstream of the flanking G-rich segment. B, template 2 is completely digested by the restriction endonuclease PacI at ambient temperature. The full-length, undigested template and the expected digestion products are indicated with arrows. C, Western blot of post-transcription Pol II complexes separated into promoter-proximal complexes (bead-bound, B) and stalled elongation complexes (supernatant, S) by PacI digestion at ambient temperature. Proteins are resolved on an 8% SDS-polyacrylamide gel, transferred to a PVDF membrane, and detected with the anti-pan-CTD antibody 4H8. D, Western blot Pol II complexes stalled on template 2 and challenged with 75 nm Rat1·Rai1 complex. The bound (B) and released (R) fractions were processed as described in C.

FIGURE 4.

Catalytically active Rat1 is able to dislodge stalled Pol II transcription complexes in vitro. A, purified recombinant exo-Rat1·Rai1 complex (left panel) is shown on an 8% SDS-polyacrylamide gel stained with silver. Purified recombinant wt Rtt103 (middle panel) and Δ-CID Rtt103 (right panel) are shown on 12% SDS-polyacrylamide gels stained with silver. B, Western blot of Pol II elongation complexes stalled on template 1 and challenged with Rat1·Rai1, exo-Rat·Rai1, or RNase A. C, Western blot of Pol II elongation complexes challenged with Rat1·Rai1 or exo-Rat·Rai1, with or without Rtt103. D, Western blot of Pol II elongation complexes challenged with exo-Rat·Rai1, with or without different forms of Rtt103. E, nascent transcripts bound to Pol II elongation complexes are incubated with the indicated recombinant proteins. RNA products, indicated by arrows, are separated on a 7 m urea/8% polyacrylamide gel. F, quantification of chemiluminescent band intensities from immunoblots such as those displayed in A, C, and D. The fraction of Pol II released from template 1 is plotted for the indicated conditions and represents an average of two to five experiments. All Western blots (B, C, and D) are performed with the 4H8 antibody.