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1.
FIG. 5.

FIG. 5. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

Functional interaction of Pta1 with Ssu72 and Ess1. Overexpression of PTA1 is lethal in the ess1H164R mutant. The wild-type (WT) (W303-1a), ess1A144T (YGD-TS8W), and ess1H164R (YGD-TS22W) strains that had been transformed with either the multicopy-number GAL1p-PTA1 plasmid (pN1681) or pYES vector (V) alone were streaked onto −Ura medium containing either 4% glucose (Glc) or 2% galactose plus 0.1% raffinose (Gal). Plates were photographed after 5 days incubation.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
2.
FIG. 3.

FIG. 3. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

The Pho, Gal, and PHO5 activation defects of ess1 mutants are suppressed by multicopy expression of SSU72 and SUA7. (A) Tenfold serial dilutions of the same strains described in the legend to Fig. 1 were spotted onto −Ura medium containing either high (1 mM) or low (5 μM) Pi concentrations or onto SC medium containing either 2% glucose (Glc) or 2% galactose (Gal). The plates were photographed after 4 days of incubation at 30°C. (B) The PHO5p-lacZ reporter plasmid was introduced into the same strains described in Fig. 1 and assayed for β-galactosidase activities under repressing (+Pi) or inducing (−Pi) conditions, as described for Fig. 2B.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
3.
FIG. 2.

FIG. 2. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

The ess1 mutants are defective for PHO5, GAL10, and INO1 transcription. (A) Pho, Gal, and Ino phenotypes associated with the ess1 mutants. Tenfold serial dilutions of wild type (WT) (W303-1a), ess1A144T (YGD-TS8W), and ess1H164R (YGD-TS22W) strains were spotted onto the indicated media. Plates were photographed after 4 days of incubation at 30°C. (B) PHO5p-lacZ, GAL10p-lacZ, and INO1p-lacZ reporter plasmids were introduced into the wild-type (W303-1a), ess1A144T (YGD-TS8W), and ess1H164R (YGD-TS22W) strains, and the resulting transformants were assayed for β-galactosidase activities under repressing (+Pi, +Glc, or +Ino) or inducing (−Pi, +Gal [without raffinose], or −Ino) conditions. All assays were performed in duplicate, using three independent transformants, and values (nmol/min/mg protein) are presented as means ± standard deviations.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
4.
FIG. 8.

FIG. 8. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

Model depicting the role of the Ess1 PPIase in CTD pSer5-Pro6 dephosphorylation. The Kin28 kinase of the general transcription factor TFIIH catalyzes Ser5 phosphorylation. Although the specificity of Kin28 for cis versus trans substrate specificity is unknown, structural constraints might limit the pSer5-Pro6 product to the cis conformation. If Ssu72 recognizes specifically pSer5-Pro6 in the trans conformation, then Ess1-catalyzed cis-trans isomerization would be required to generate the Ssu72 substrate. This model is consistent with the trans specificity of several mammalian Ser/Thr-Pro kinases and phosphatases and with the specificity of the Pin1 PPIase (57). CprX denotes a cyclophilin A PPIase, which might be required to catalyze cis-trans isomerization of unphosphorylated Ser5-Pro6, a possibility suggested by suppression of ess1 by multicopy expression of CPR1, one of the eight cyclophilin A genes in yeast (56). The “c” and “t” subscripts denote the cis and trans conformations of the peptidyl prolyl bond.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
5.
FIG. 6.

FIG. 6. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

The ess1 mutations affect Pol II CTD Ser5 phosphorylation in vivo. The wild-type (WT) (W303-1a), ess1A144T (YGD-TS8W), and ess1H164R (YGD-TS22W) strains that had been transformed with either the high-copy-number pRS423 vector (V) or the same vector carrying the SSU72 gene (no. 72), were grown in −His selective medium to the optical density at 600 nm of 0.7 to 0.8. Total cell extracts were then analyzed by Western blotting using antibodies specific to the Ser5 phosphorylated form of the CTD (H14), hypophosphorylated CTD (8WG16), Rpb3, Ssu72, Ess1, or the Rpa1 protein (loading control). The Ssu72 antibody detects a doublet, with the upper band corresponding to phosphorylated Ssu72 (B.-S. Chen and M. Hampsey, unpublished results).

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
6.
FIG. 1.

FIG. 1. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

Genetic interactions among Ess1, Ssu72, and TFIIB (SUA7). (A) Suppression of ess1 temperature-sensitive growth defects by multicopy expression of SSU72 and SUA7. Tenfold serial dilutions of isogenic wild-type (WT) (W303-1a), ess1A144T (YGD-TS8W), and ess1H164R (YGD-TS22W) strains that had been transformed with the multicopy-number (2μm) vector alone (YEp24), or its derivatives carrying either SSU72 (pM1402) or SUA7 (pM264), were spotted onto −Ura medium. Plates were photographed after 2 (30°C) or 3 (37°C) days of incubation. (B) Synthetic lethality associated with ess1H164R and specific sua7 alleles. Plasmid shuffle strains YMH1076 (WT) and YMH1077 (ess1H164R) carrying plasmid pM269 (SUA7-URA3) and HIS3 plasmids with the indicated sua7 allele were spotted in duplicate on either −His −Ura medium or 5-FOA medium, which counterselects pM269. The sua7 alleles encode single-amino-acid replacements and were described previously (54). (C) Synthetic lethality associated with ess1 and ssu72 mutations. Plasmid shuffle strains YMH1036 (WT), YMH1040 (ess1A144T), and YMH1044 (ess1H164R) carrying plasmid pM586 (SSU72-URA3) and TRP1 plasmids with the indicated ssu72 allele were spotted in duplicate on either −Trp −Ura medium or 5-FOA medium, which counterselects pM586.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
7.
FIG. 4.

FIG. 4. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

Depletion of Ess1 is not detrimental to pre-mRNA 3′-end processing in vitro. (A) Western blot analysis of cell extracts from the wild-type (WT) strain (W303-1a) or Ess1-TAP strain (H-351), either prior to (+) or following (−) Ess1-TAP depletion. The blot was probed with either Ess1 antibody (α-Ess1) or anti-PAP (α-PAP), which detects the TAP tag. (B) For the coupled cleavage-polyadenylation assay (left), the extracts described in panel A were incubated with ATP and 32P-labeled full-length GAL7-1 RNA (Pre) (black and white rectangle) for 20 min at 30°C. The same conditions were used for poly(A) addition assays (right), except that the precleaved GAL7-9 RNA was used as substrate (black rectangle). Products [An, poly(A) RNA] were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. Positions of substrates and products are indicated on the left side of each panel.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.
8.
FIG. 7.

FIG. 7. From: Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries .

Ess1 cross-links to the promoter and terminator regions of the PMA1 and PHO5 genes. (A) Schematic depiction of the PMA1 gene showing the position of its promoter (black box) and two polyadenylation sites (vertical arrows). The regions probed by ChIP are denoted 1 to 9. (B) ChIP analysis of Ess1 cross-linking to PMA1 using strain H-351 (Ess1-TAP). ChIP was done using IgG-agarose beads. P1 to P4 and P6 to P9 correspond to regions 1 to 4 and 6 to 9 in panel A; lane V denotes a nontranscribed region of chromosome V that serves as an internal background control. All PCR primer pairs used in this analysis are identical to those described previously (27). The input signal represents DNA prior to immunoprecipitation. (C) Quantification of the data shown in panel B. (D) Schematic depiction of the PHO5 gene. The regions probed by ChIP are denoted A to D. Region D encompasses the PHO5 poly(A) site (22). (E) ChIP analysis of Ess1-TAP and Rpb3 cross-linking to PHO5 using the same strains and antibody as described for panel B, under repressing (+Pi) and activating (−Pi) conditions, as described for Fig. 3. (F) Quantification of the data shown in panel E.

Shankarling Krishnamurthy, et al. Mol Cell Biol. 2009 June;29(11):2925-2934.

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