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1.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 2. Identity of ts and lethal substitutions in Ess1. (A) Alignment of yeast Ess1 and homologs from Drosophila (Dodo) and human (Pin1), showing the position (underlined) and identity of the substitutions. (B) Model of the Ess1 structure based on X-ray crystallographic data of Pin1 (Ranganathan et al., 1997) showing the location of selected mutations. (C) Model of the active site of wild-type Ess1, C120R and H164R mutants, each containing a bound phosphoserine–proline dipeptide in the cis configuration. Although there are minor rearrangements, none appears to preclude binding of the substrate peptide.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
2.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 7. Model for the action of Ess1 in transcription. Ess1 binds the phosphorylated CTD of RNA pol II, catalyzing its isomerization, thus acting as a regulatory switch for loading of proteins required for initiation, elongation, termination and 3′ end formation. In this model, Ess1 coordinates the sequential steps of transcription by changing the three-dimensional structure of the CTD, altering the affinity of protein–CTD interactions. Binding of Ess1 to the CTD would be regulated by phosphorylation–dephosphorylation by CTD kinases and CTD phosphatases (e.g. Fcp1). Ess1 might instead work stoichiometrically; Ess1 would sterically block (or promote) binding of RNA pol II-associated proteins to the phosphorylated CTD.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
3.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 5. ESS1 shows a genetic interaction with RPB1. (A) The ess1H164R mutant is synthetically lethal with a reduced dosage of the RPB1 gene. Cells of the indicated genotype (W303-1A background) were streaked to rich medium and grown for 3 days at permissive temperature (30°C). (B) The ess1H164R mutant is synthetically lethal with reduced levels of RPB1 expression. Cells of the indicated genotype (W303-1A background) were streaked to selective medium and grown for 3 days at permissive temperature (25°C). pRPB1 is a CEN-ARS (URA3) plasmid that expresses the large subunit of RNA pol II from its natural promoter. PLEU-RPB1 is a CEN-ARS (TRP1) plasmid (also known as pLEU-RPO21; Archambault et al., 1996) that expresses the large subunit of RNA pol II from the repressible LEU2 promoter. The plate shown contains 2 mM leucine, threonine and isoleucine, which represses the PLEU promoter, driving low-level expression of RPB1.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
4.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 3. High-copy suppressors of the lethal ess1 mutant phenotype. (A) Suppression of ess1H164R host cells at 37°C by six different high-copy plasmids carrying the indicated genes. Control plasmids were YEpESS1 or an empty vector (pRS426). CaRPB7 is from Candida albicans. Cells were streaked onto medium that lacked uracil and incubated at 37°C for 5 days. (B) Fcp1 phosphatase activity is required for suppression of ess1ts mutants. Suppression of ess1H164R mutant cells at 37°C was tested using high-copy plasmids (2µ; TRP1) encoding wild-type (FCP1) or mutants alleles (fcp1-1 and fcp1-2) that lack CTD phosphatase activity (Kobor et al., 1999). (C) Western analysis to detect cyclophilin A (Cpr1) in whole-cell lysates from ess1H164R mutant cells carrying the indicated suppressors, or control plasmids, YEpESS1 or pRS426. Cells were grown at permissive temperature and shifted to 37°C for 4 h. Rabbit anti-cyclophilin A serum was used at a 1:3000 dilution.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
5.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 1. Growth properties of ess1ts mutants. (A) Growth of wild-type (W303-1A) and selected mutants in rich medium (YEPD) at permissive and non-permissive temperatures. The ess1W15R allele was on a centromeric plasmid in an ess1Δ background. The ess1L94P and ess1H164R alleles were integrated into the genome. (B) Upper panel: growth of strains containing integrated ess1ts alleles. Cells were grown to mid-log phase in YEPD medium, and serial 1:5 dilutions were spotted onto plates and incubated for 1–2 days at the indicated temperatures. Lower panel: growth of strains carrying plasmid-borne (CEN) copies of ess1ts alleles in an ess1Δ host. Cells were grown and spotted as in the upper panel. (C) Mitotic arrest and nuclear fragmentation of ess1ts mutants. Cells (ess1H164R) were grown to mid-log phase and shifted to 37°C for 8 h. Cells were fixed, stained with DAPI, and visualized under Nomarski optics (DIC) or UV light (DAPI). (D) Western analysis of mutant proteins. Cells were grown at permissive temperature and shifted to 37°C for 4 h prior to harvesting. Equal amounts of total cell lysates were analyzed. Rabbit anti-Ess1 antiserum was used at a 1:4000 dilution.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
6.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 4. Ess1 interacts with the CTD of RNA pol II in vivo and in vitro. (A) A schematic of the two-hybrid experiment is shown on the left. Yeast cells expressing the indicated proteins were streaked on an X-gal-containing plate and incubated overnight at 30°C (right). The LexA1–87–CTD bait protein contains nine repeats of the YSPTSPS motif and interacts with activation domain-tagged Ess1 but not with the control protein, Bicoid. Neither LexA1–87–CTD nor Ess1 alone activates transcription. (B) Ess1, but not cyclophilin A, interacts with LexA1–87–CTD in vitro. Cyclophilin A- (CypA), Ess1- or FKBP12-conjugated affinity beads or control beads were reacted with total protein extracts from cells expressing either a LexA1–87–CTD fusion protein or intact LexA1–202. Bound proteins were eluted and analyzed by western blotting with LexA antisera. Extract denotes whole-cell lysates not reacted with affinity beads. (C) Far-western analysis shows that Ess1 reacts with a protein that co-migrates with hyperphosphorylated RNA pol II. Lanes 1 and 2 are marker proteins and Coomassie-stained total yeast extracts, respectively. Lanes 4 and 7 are far-western samples using His-tagged Ess1 or CypA proteins as probes, and detected using the cognate antibodies. Lanes 3, 5, 6 and 8 are standard western blots reacted with the indicated antisera.

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.
7.

Figure. From: The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

Fig. 6. Selective effects on transcription in ess1 mutant cells. (A) LacZ reporter gene expression in yeast. Left: wild-type or ess1ts mutant yeast were transformed with a plasmid expressing LexA–Gal4 (pSH17-4; Hanes and Brent, 1989), and a 4×-LexA operator-lacZ reporter construct (pSH18-8; S.D.Hanes and R.Brent, unpublished) at 23°C. β-galactosidase activity was measured in liquid cultures of five independent isolates. Similar results were obtained at 37°C (not shown). Right: cells were transformed with a Bicoid-site lacZ reporter (pWZ11-1; Zhu and Hanes, 2000) and treated as above, except that cells were incubated at 35°C. The differences between wild-type and the ess1 mutants were less pronounced at 23°C (not shown). (B) Northern analysis of genes encoding mitotic regulators in ess1H164R mutant cells. Wild-type cells or ess1H164R mutant cells with control vector pRS426 (–) or the indicated multicopy suppressors were grown in selective medium and shifted to 37°C for 4 h prior to harvesting. A 6 µg aliquot of total RNA was loaded per lane. Duplicate blots were hybridized with random primed 32P-labeled probes for CPR1 (cyclophilin A) and MIH1 (Cdc25 phosphatase), or CLB2 (cyclin B) and HHF2 (histone H4).

Xiaoyun Wu, et al. EMBO J. 2000 July 17;19(14):3727-3738.

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