1. Volume 45, Issue 2, Pages 158-170 (January 2012)
A Universal RNA Polymerase II CTD Cycle Is Orchestrated by Complex Interplays between Kinase, Phosphatase, and Isomerase Enzymes along Genes 
Alain R. Bataille, Célia Jeronimo, Pierre-Étienne Jacques, Louise Laramée, Marie-Ève Fortin, Audrey Forest, Maxime Bergeron, Steven D. Hanes, François Robert 
Molecular Cell 
Volume 45, Issue 2, Pages (January 2012)
DOI: /j.molcel
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2. Figure 1 A Uniform CTD Cycle at Most Transcribed Genes
(A) Profile of RNAPII using either anti-CTD (8WG16), anti-myc (9E10; Rpb1-9myc), or anti-Rpb3 (W0012) antibodies on a metagene made with long (1500–2500 bp) and transcribed genes (based on 8WG16 average binding across the open reading frame [ORF]).
(B) Profile of RNAPII (8WG16) and its phosphorylation marks (P-Ser2 [H5 and 3E10], P-Ser5 [H14 and 3E8], P-Ser7 [4E12]) along metagenes representing three classes of genes based on average gene length (see Experimental Procedures for details). Profiles are shown for long (2,000 +/− 500 bp; solid line), medium size (1,000 +/− 250 bp; dashed line) and small (500 +/− 100 bp; dotted line) genes.
(C–E) Genomic regions harboring genes that show canonical (Box1 and Box2) and noncanonical (Box3–Box5) CTD phosphoserine profiles. Shaded boxes (Box1–Box5) highlight regions described in the text. Annotated genes are shown in gray and the nonannotated usURA8 cryptic unstable transcript (CUT) is shown in black. ChIP-chip enrichment ratios are shown for all RNAPII antibodies tested in this study in wild-type cells. See also Figure S1.
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3. Figure 2 Complex Interplays between CTD Kinases
(A–C) Profile of RNAPII and its phosphoisoforms along a metagene corresponding to long and expressed genes in A) kin28-as (dashed trace) and its isogenic wild-type (solid trace); B) ctk1Δ (dashed trace), bur2Δ (dotted trace) and their isogenic wild-type (solid trace); C) kin28-as (solid trace) and kin28-as/bur2Δ (dotted trace). The top panel shows total RNAPII (Rpb3, W0012) and the panels below show RNAPII (8WG16), P-Ser5 (3E8), P-Ser7 (4E12) and P-Ser2 (H5, 3E10), all normalized for the difference in RNAPII (Rpb3, W0012) level in the mutants (as described in Supplemental Experimental Procedures). This allows one to appreciate the difference in phosphorylation levels without being biased by the fact that RNAPII does accumulate at the 5′end of genes in some mutants. See also Figure S2.
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4. Figure 3 Ssu72 Dephosphorylates Ser5 and Ser7 at the End of Genes
(A) Profile of Ssu72 (3myc-Ssu72) along a metagene with the polyadenylation factor Pti1 (Pti1-3myc) as a control.
(B) Profile of RNAPII (8WG16) and its phosphoisoforms (3E8, 4E12, H5, and 3E10) along a metagene in the degron-ssu72 strain (dashed traces) and its isogenic wild-type (solid traces). Only the 3′end of genes is shown. See Figure S3 for the complete gene.
(C) In vitro CTD phosphatase activity of recombinant Ssu72. GST-CTD substrate was hyperphosphorylated using Kin28- and Ctk1-TAP complexes as described in Experimental Procedures. Increasing amounts (0.5, 2.5, and 5 μM) of GST-Ssu72 (lanes 3–5) were incubated with the hyperphosphorylated GST-CTD (P-CTD). An equivalent amount (5 μM) of a catalytic mutant GST-Ssu72 C15S was used as a negative control (lane 6). Phosphatase reactions were stopped by the addition of gel-loading buffer, resolved on SDS-PAGE gels, and subjected to western blotting with the indicated antibodies. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Ess1 Is Required for the Dephosphorylation of Ser5 and Ser7 at the End of Genes
Profile of RNAPII (8WG16) and its phosphoisoforms (3E8, 4E12, and 3E10) along a metagene in the ess1-H164R strain (dashed traces) and its isogenic wild-type (solid traces). Only the 3′end of genes is shown. See Figure S4 for the complete gene.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Two Variants of P-Ser2 Are Removed in Two Steps by the Concerted Action of Fcp1 and Ssu72
(A) In vitro CTD phosphatase activity of TAP-purified Fcp1. The experimental diagram is indicated at the top of the panel. GST-CTD substrate was hyperphosphorylated using Kin28- and Ctk1-TAP complexes. Increased amounts of Fcp1-TAP complex (lanes 3–5) were incubated with either fully phosphorylated GST-CTD substrate or with an hyperphosphorylated GST-CTD that was first treated with 5 μM of GST-Ssu72 (lanes 8–10) or 5 μM of a catalytic mutant GST-Ssu72 C15S (lane 12). An eluate from an untagged strain was used as a control of the Fcp1-TAP phosphatase activity (lanes 6 and 11). Phosphatase reactions were stopped by the addition of gel-loading buffer, resolved on SDS-PAGE gels, and subjected to western blotting with the indicated antibodies.
(B) Profile of RNAPII (W0012, 8WG16) and P-Ser2 (H5, 3E10) along a metagene in wild-type (solid traces) and fcp1-1 (dashed traces) cells. The gene lists contributing to the metagenes have been selected based on anti-Rpb3 (W0012) occupancy in each strain. The P-Ser2 profile is normalized based on 8WG16 occupancy in order to correct for CTD masking. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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