Ccr4–Not is recruited to genes during elongation by RNAPII. (A) Schematic of RNR3 and primer locations. (B–D) ChIP analysis of Dhh1-myc, Ccr4-myc, and Not5-myc across RNR3 in cells untreated or treated with MMS for 2.5 h. Background (untagged) percent immunoprecipitated (%IP) was subtracted from anti-myc %IP. %IP was calculated by dividing the immunoprecipitation DNA signal by the Input DNA signal using each primer set. POL1 was used as a control. (E) Schematic of GAL1 and primer locations. (F) Recruitment of Dhh1-myc and Ccr4-myc to the GAL1 ORF under dextrose or galactose conditions. (G) Location of Dhh1p at GAL1 in cells grown in raffinose or galactose media. (H) Cells were grown overnight in raffinose and induced with 2% galactose for 15, 30, 60, and 90 min. RNAPII (dotted line) and Dhh1 (solid line) densities at the GAL1 ORF. (I) Galactose-grown cells were treated with 4% dextrose and cross-linked at 2, 5, 10, and 15 min.

Ccr4–Not associates with RNAPII. (A) Coimmunoprecipitation of myc-tagged Ccr4–Not subunits with RNAPII. Whole-cell extracts (WCEs) were either treated or left untreated with 100 μg/mL RNase A at room temperature prior to addition of antibody. Rpb1 subunit of RNAPII (8WG16) was detected by Western blotting. The amount of immunoprecipitated protein recovered was analyzed by Western blotting using an anti-myc antibody. Since Ccr4–Not subunits run at different molecular weights, regions corresponding to the location of each myc-tagged protein were cut from their respective regions on the membrane and placed in a row. (B) Same as in A except immunoprecipitation was performed using a polyclonal Dhh1 antisera. Anti-Dhh1 was used to probe the blot as an immunoprecipitation control. (C) Same as in B except extracts from various transcription factor mutants were analyzed.

The Ccr4–Not complex interacts directly with yeast RNAPII elongation complexes. (A) Silver-stained SDS-PAGE gels showing the composition of yeast RNAPII purified from a TAP-Rpb4 strain (left) and the Ccr4–Not complex purified from a TAP-Not4 strain (right). (B) Schematic representation of the in vitro elongation system. RNAPII initiates transcription with UpG on the tailed template and stalls at the G-residues located at the end of the G-less cassette. (C) Native gel analysis of the interaction of Ccr4–Not with elongation complexes. RNAPII elongation complexes were formed on the tailed template (EC70) containing a 70-nt radiolabeled nascent transcript. One-hundred nanograms of template was used in each reaction. Approximately 100 ng (∼0.25 pmol) of RNAPII was present in each sample. Elongation complexes stalled at the end of the G-less cassette were incubated for 10 min with increasing amounts of purified Ccr4–Not complex (∼0.5, 1, and 1.5 pmol of Ccr4–Not complex). The concentrations of all proteins were estimated by comparing intensities of their bands with those of known amounts of BSA on a silver-stained SDS-PAGE gel. RNAPII-only lane contains 1 μg of BSA. (D) Yeast (yRNAPII), Drosophila (dRNAPII), and archaeal polymerase from Pyrococcus furiosus (aPol) were used to generate elongation complexes (EC70) on the tailed template. Ccr4–Not complex (0.5 pmol and 1 pmol) was added to each of the elongation complexes and reactions were analyzed on a 4% native gel.

Ccr4–Not stimulates the resumption of transcription from arrested RNAPII elongation complexes. (A) Outline of the in vitro elongation runoff assay. Arrested RNAPII elongation complexes (EC70) were formed in the absence of GTP. RNAPII is able to resume elongation and generate a 150-base runoff transcript after the addition of GTP and UTP. (B) In vitro runoff assay in the presence of Ccr4–Not. Arrested elongation complexes were incubated with 1.5 pmol of Ccr4–Not complex or 1 μg BSA for 10 min, and then GTP and UTP were added to allow runoff. A total of 1.5 pmol of complex was used because it is just above the amount required to fully shift polymerase in the gel shift assay (Fig. 3). (C) TFIIS rescues arrested EC70 complexes. TFIIS (0.5 pmol) was added to the reaction after forming the arrested EC70. (D,E) Ccr4–Not, unlike TFIIS, does not stimulate the nucleolytic activity of RNAPII. In vitro elongation system was set up as described above, with the exception that O-me GTP was added to form the arrested elongation complex. Prior to the addition of nucleotides, 0.5 pmol of TFIIS (D) or 1.5 pmol of Ccr4–Not complex (E) was added to the reactions.

Ccr4–Not functions in a transcript length-dependent manner and binds the transcript. (A,B) Transcription elongation assays from EC31 and EC22 complexes. Assay conditions and experimental design are described in the legend for Figure 4. (C) Native PAGE of EC complexes. The assay was carried out as described in Figure 3C, except that Ccr4–Not was added at 1:1 and 2:1 to RNAPII. (D) UV cross-linking of Ccr4–Not to the transcript in elongation complexes. EC complexes were formed from templates containing a 70-, 31-, and 22-nt G-less cassette, forming EC70, EC31, and EC22, respectively. Transcription was carried out in the presence of radiolabeled CTP and bromouridine. After the formation of the ECs, Ccr4–Not was added to the complexes and the mixture was cross-linked by UV light as described in the Materials and Methods. DNase and RNase were added prior to electrophoresis. The migration of molecular weight markers (in kilodaltons) are indicated on the left, and the Ccr4–Not-specific band is marked by a star on the right.

Ccr4–Not promotes elongation in vivo. (A) Schematic of GAL1_P-YLR454W and primer locations. (B) ChIP analysis of RNAPII density across GAL1_P-YLR454W in wild-type and mutant strains under galactose-inducing conditions. RNAPII%IP in mutant strains was normalized to the corresponding region in wild type, and the promoter region for each strain was then set to 1. (C) ChIP of relative RNAPII density for wild-type and mutant strains at the 2-kb region under inducing and repressing conditions for the indicated times. RNAPII density in galactose-grown cells was set to 1, and time points after addition of glucose were normalized to the RNAPII%IP prior to repression. (D) ChIP of relative RNAPII density in wild-type and ccr4Δ strains following 2 min of treatment with dextrose. A ratio of (RNAPII%IP at 2 min + glucose)/(RNAPII%IP in galactose) was calculated for wild-type and ccr4Δ strains at the indicated primer regions.

Model for the rescue of arrested elongation complexes by Ccr4–Not. (Top) Transcription blocks lead to arrest and backtracking of polymerase. The 3′ end of the transcript is out of register with the active site (yellow starburst), preventing productive elongation. (Middle) Transient forward excursions of polymerase threads transcript out of the RNA exit channel, which can associate with Ccr4–Not. (Bottom) Cycles of transcript binding and release by Ccr4–Not during forward excursions promote elongation by locking RNAPII into an elongation-competent form.