1. The Yeast Capping Enzyme Represses RNA Polymerase II Transcription
Lawrence C. Myers, Lynne Lacomis, Hediye Erdjument-Bromage, Paul Tempst 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1
Characterization of a General Repressor of RNA pol II Transcription Antagonized by RNA in Yeast Whole-Cell Extracts
(A) Chromatographic scheme for initial fractionation of WCE, and identification of a general repressor and its antagonist (factor C).
(B) Activities of the Hep20 fraction and the RNA isolated from the Hep20 fraction in transcription. Transcription was performed with templates containing binding sites for Gal4 (GAL4:CG-) and Gcn4 (GCN4:CG-) upstream of the Saccharomyces cerevisiae CYC1 promoter fused to a G-less cassette (Myers et al., 1998). All reaction mixtures contained the DE400 fraction (35 μg total protein), 50 ng of TBP, 50 ng of TFIIB, 10 ng of Gal4-VP16, and varying amounts of the Hep20 fraction or the purified RNA from the Hep20 fraction.
(C) Activity of the partially purified general repressor with highly purified transcription factors. Transcription was performed with the two template system as described above. All reaction mixtures contained purified Mediator and basal transcription factors as previously described (Myers et al., 1998), and 10 ng of Gcn4. The partially purified general repressor (Mono-Q fraction 28; 800 ng of protein) and the poly (A) (Sigma) antagonist (62 ng) were added to the reactions as shown. Quantification of the Gcn4-activated transcript using a phosphorimager revealed that the addition of the repressor resulted in a 5.8-fold decrease in transcription.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
The General Repressor Functions at Reinitiation in Transcription Reactions Reconstituted from Purified Factors
(A) Method used to separate the contribution of initiation and reinitiation in transcription reactions. Mediator, basal transcription factors, the activator Gcn4, the DNA templates (as described in Figure 1C), and various amounts of the general repressor (Mono-Q fraction 28) were added at t1. Preinitiation complexes were formed during a 20 min incubation at room temperature. The addition of CTP and UTP at t2 was followed by a variable reaction time that was stopped at t4.
(B) Transcription reactions in the presence and absence of sarkosyl. Three minutes was allowed after the addition of NTPs for the elongating polymerase to become resistant to sarkosyl (Coda-Zabetta and Boam, 1996). Sarkosyl was added at t3 to a final concentration of 0.125% w/v, and reactions were allowed to proceed for 60 min. Poly (A) (62 ng/rxn) was added to selected reactions at t3 to antagonize the repressor and calculate the potential for reinitiation in each reaction mixture. Reactions were analyzed as described in Figure 1C, but only the products of the GCN4:CG- template are shown here.
(C) Increasing the amount of the general repressor results in selective inhibition of reinitiation. The transcription reactions shown in (B) were quantified using a phosphorimager. The amount of activated transcription arising from initiation (RNA pol II complexes formed during the preincubation) was measured as the amount of transcription from the GCN4 template in the presence of sarkosyl. The difference in initiation with increasing amounts of general repressor is described as a percentage of the signal in the absence of repressor. The amount of activated transcription arising from reinitiation was measured as the difference between the total amount of transcription and the amount of transcription in the presence of sarkosyl from the GCN4 template. The difference in reinitiation with increasing amounts of general repressor is described as a percentage of the reinitiation signal in the absence of repressor. The expected or “100%” reinitiation in each reaction with the Mono-Q fraction is normalized according to the total amount of transcription with poly (A) present.
(D) Time course analysis of transcription reactions. Transcription reactions were performed and analyzed as described in (A) and (B) with the following modifications: no sarkosyl is added to any of the reactions, all reactions with the repressor activity contain 0.9 μg Mono-Q fraction, and the reaction time (t2 to t4) is varied between 4 and 80 min.
(E) Rate of multiround transcription in the presence and absence of repressor activity. The transcription reactions shown in (D) were quantified using a phosphorimager. The total amount of transcription is plotted as a function of the length of the reaction time.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 Purification of the Repressor of Transcriptional Reinitiation
(A) Chromatographic scheme used to purify the repressor. Repression of transcription in a purified system, which was antagonized by the addition of poly (A), was the criteria used at each stage to analyze and pool fractions.
(B) SDS-PAGE analysis of purified repressor. Approximately 200 ng of protein was analyzed by SDS-PAGE on a 10% gel stained with colloidal Coomassie (Invitrogen). The two bands (indicated by the arrows) were excised, subjected to mass spectrometry analysis, and identified as the yeast Ceg1 and Cet1 proteins as shown.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Repression of Transcriptional Reinitiation by Recombinant Capping Enzymes
(A) Purification of recombinant capping enzymes. SDS-PAGE analysis of E. coli expressed 6-histidine tagged and purified full-length rCeg1p and truncated rCet1p ( ). Proteins are revealed by Coomassie staining.
(B) Inhibition of activated transcription in the purified system by the native purified repressor (pool of Phenyl Hi-Trap fractions, ≅100 fmol capping enzyme/reaction), recombinant rCeg1 ([A], 400 fmol/reaction), rCet1 ([A], 200 fmol/reaction), and rCet1/rCeg1 (200 fmol/reaction). The purified transcription system, as described in Figure 1C, was used to assess the ability of the native and recombinant proteins to repress transcription. The fold inhibition is measured for the Gcn4p-activated transcription signal and is fully reversible by the addition of poly (A).
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 Depletion of Capping Enzyme Results in Derepression
(A) Western analysis of capping enzyme depletion from DE400 fraction. A crude DE400 fraction was incubated with anti-Cet1p antibodies immobilized on protein-A beads, and the supernatant was recovered. An equal volume of load (DE400) and supernatant (DE400[ΔCet1/Ceg1]) was analyzed by Western blotting. The capping enzyme (Cet1, Ceg1) is depleted from the DE400 fraction in comparison to RNA pol II (Rpb1) and Mediator (Med4, Med7).
(B) Transcription in a DE400 and DE400(ΔCet1/Ceg1) fraction. In vitro transcription assays were performed as described in Figure 1B, with the exception that the activator Gcn4p was used in place of Gal4-VP16. The DE400(ΔCet1/Ceg1) was isolated as described in (A), and the DE400 was mock depleted using uncoupled protein-A beads. Recombinant capping enzyme complex (rCet1[ ]/Ceg1) was added back to the indicated reactions as described in Figure 4. The indicated reactions contain 2.5 μg of poly (A) RNA.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Inactivation of Cet1p Increases the Density of Polymerases on the lacZ Gene In Vivo
(A) Run-on transcription assay of Gal5-CYC1-lacZ gene on a multicopy plasmid in the mutant cet1-438 ts, cet1-401 ts, and wild-type (CET1) strains at 30°C and 37°C in the absence (−) and presence (+) of galactose (GAL). Cells were grown for 3 hr in 2% galactose and harvested 60 min after shifting to 37°C. Since differences in the length, number of U residues, and total amount of the lacZ-5′, lacZ-3′, and rRNA probes were not accounted for, only differences in transcription between the same probe under different conditions can be directly compared. The blots are exposed to account for normalization of the rRNA probe as a loading control.
(B) Plot of fold change in run-on transcription upon shifting from 30°C to 37°C. Using a phosphorimager, the transcription signal hybridized to the lacZ-5′ and lacZ-3′ probes, as shown in (A), was quantified and normalized using the rRNA signal as a loading control, and the ratio of signal obtained at 37°C to signal obtained at 30°C was plotted for both probes in the absence and presence of galactose.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7 Model of Capping Enzyme Repression of Reinitiation
This schematic model for capping enzyme-dependent inhibition of transcriptional reinitiation is based on the model of Hahn and colleagues (Yudkovsky et al., 2000) for preinitiation and reinitiation. The dashed circle around TFIIE reflects that it may be an unstable scaffold component (Yudkovsky et al., 2000). The shading of TFIIF in the elongation complex reflects recent evidence (Pokholok et al., 2002) that TFIIF may not be present in this complex. Specific inhibition of reinitiation by the capping enzyme suggests that the scaffold may exist in two forms: a blocked scaffold that is unable to undergo reinitiation and an open scaffold that is competent for reinitiation. RNA pol II is not limiting in our reactions, so it is unlikely that the capping enzyme/CTD-P interaction is titrating out pol II. Several mechanisms, including capping enzyme binding to, or release from, the CTD, could serve as a means (marked by a “?” in the figure) to displace capping enzyme inhibition and permit reinitiation.
Molecular Cell  , DOI: ( /S (02) )