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1.
Figure 5

Figure 5. hERK5 and hPAF1 interact to drive FKS2 expression. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) and (B) RT-PCR of FKS2. (A) Expression of either hPAF1, or hPAF1-AA in a paf1Δ mutant (DL3792). (B) Co-expression of hERK5 and hPAF1 in a paf1Δ mpk1Δ mlp1Δ mutant (DL3980). WCE; whole-cell extract.

Ki-Young Kim, et al. Cell. ;144(5):745-756.
2.
Figure 7

Figure 7. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

Model for Mpk1-driven FKS2 transcription. (A) Under non-inducing conditions, weak transcription initiation is attenuated by association of the Sen1-Nrd1-Nab3 complex to the elongation complex. (B) Under inducing conditions, activated Mpk1 recruits Swi4-Swi6 to the FKS2 promoter; (C) Pol II and the Paf1C are recruited to the promoter in a Swi6-dependent manner; (D) Mpk1 associates with Paf1, possibly by hand off from Swi4. Mpk1 functions as an anti-termination factor by blocking recruitment of the termination complex.

Ki-Young Kim, et al. Cell. ;144(5):745-756.
3.
Figure 4

Figure 4. The paf1-4A mutant is blocked for transcription elongation of FKS2, but not CLN2. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) RT-PCR of FKS2 and CLN2. A paf1Δ strain (DL3792) expressing PAF1, paf1-4A, or bearing empty vector was subjected to heat stress. Total RNA was processed for RT-PCR detection of FKS2 and CLN2 mRNA and 18S RNA. WCE; whole-cell extract. (B–F) ChIP analyses in a paf1Δ strain (DL3548) expressing the indicated forms of Paf1. (B) Top; schematic of the CLN2 gene showing regions amplified for ChIP along the length of the gene. P, promoter; M, middle coding region; C, C-terminal coding region. Bottom; Paf1-HA ChIP on CLN2. (C) Rpb3-HA ChIP on CLN2. (D) Rpb3-HA ChIP on FKS2. (E) Paf1-HA ChIP on FKS2. (F) Mpk1-HA ChIP on FKS2.

Ki-Young Kim, et al. Cell. ;144(5):745-756.
4.
Figure 1

Figure 1. Mpk1 and Mlp1, associate with the FKS2 promoter and coding region, but Swi4 and Swi6 bind only to the promoter. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) Top; schematic of the FKS2 gene showing regions amplified for ChIP analyses along the length of the gene. P, promoter; N, N-terminal coding region; M, middle coding region; C, C-terminal coding region. Bottom; Mpk1 ChIP on FKS2. HA-tagged forms of Mpk1, an activation defective phosphorylation site mutant (Mpk1-TAYF), or a catalytically inactive form (Mpk1-K54R) were expressed from plasmids in an mpk1Δ mlp1Δ strain (DL3327). Cells were exposed to heat stress at 39°C for 2h, or unstressed at RT. A region from the middle of the DYN1 coding region was used as a negative control. (B) Mlp1 ChIP on FKS2. HA-tagged Mlp1 was expressed from a plasmid in an mpk1Δ mlp1Δ strain (DL3327). (C) Swi4 ChIP on FKS2. HA-tagged Swi4 was expressed from a plasmid in a swi4Δ strain (DL3145). (D) Swi6 ChIP on FKS2. HA-tagged Swi6 was expressed from a plasmid in a swi6Δ strain (DL3148).

Ki-Young Kim, et al. Cell. ;144(5):745-756.
5.
Figure 2

Figure 2. Requirements for association of Mpk1 with Paf1. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) The Mpk1-Paf1 interaction is stimulated by cell wall stress. Paf1-Flag was tested for co-immunoprecipitation (IP) of Mpk1-HA activated by various cell wall stresses for 2h – U, unstressed (RT); HS, heat stress at 39°C; CR, 50μg/ml Congo red; CW, 40μg/ml calcofluor white – in an mpk1Δ mlp1Δ (DL3183) strain expressing differentially tagged Mpk1 and Paf1. (B) The Paf1-Mpk1 interaction requires activating signal to Mpk1, but not catalytic activity. Mpk1-HA, an activation defective phosphorylation site mutant (Mpk1-TAYF-HA), or its catalytically inactive form (Mpk1-K54R-HA), was co-expressed with Paf1-Flag in an mpk1Δ mlp1Δ strain (DL3183). (C) The Paf1-Mpk1 association requires Swi4 and Swi6, but not Rlm1. Yeast strains were: wild-type (DL3187), swi4Δ (DL3405), swi6Δ (DL3233), and rlm1Δ (DL3586). (D) Neither Pol II, nor Paf1 are recruited to the FKS2 gene in the absence of Swi6. Rpb3-HA and Paf1-HA ChIP on FKS2 in wild-type cells (DL3187) and a swi6Δ strain (DL3233). The ADH1 promoter was used as a reference control.

Ki-Young Kim, et al. Cell. ;144(5):745-756.
6.
Figure 6

Figure 6. The Mpk1-Paf1 interaction prevents Sen1-mediated premature termination. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) RT-PCR of the FKS2 5′ region. A paf1Δ strain (DL3977) expressing PAF1, or paf1-4A was processed for RT-PCR detection of FKS2 transcripts. One primer pair detects only extended mRNA (−564 to +48). A second pair detects both attenuated transcripts and extended mRNA (−564 to −243). (B) RT-PCR of FKS2. A SEN1 paf1Δ strain (DL3977) and a sen1-1 paf1Δ strain (DL3978) expressing PAF1, paf1-4A, or bearing empty vector, were processed for RT-PCR detection of FKS2 expression after heat stress. (C) Rpb3-HA ChIP on FKS2. The same strains as in (B), but expressing Rpb3-ZZ-HA, were processed for ChIP after heat stress. V; vector. (D) Sen1-ZZ ChIP on FKS2 in a paf1Δ strain expressing Sen1-CBD-ZZ (DL3979) and the indicated forms of Paf1. (E) A region of the FKS2 promoter functions as a Sen1-dependent terminator. The indicated region of FKS2 sequence was inserted in both orientations into the ACT1 intron of an ACT1-CUP1 fusion plasmid, which was transformed into SEN1 cup1Δ (DL3990) and sen1-E1597K cup1Δ (DL3995) strains. The SNR13 terminator was used as a positive control. Serial dilutions were spotted onto plates with 0.4 mM CuSO4 and incubated for three days at the semi-permissive temperature for sen1-E1597K of 30°C. (F) The Nab3-binding site at position −476 in FKS2 is responsible for transcriptional dependence on the Mpk1-Paf1 interaction. Expression of the indicated FKS2-lacZ fusion was measured before and after heat stress in a paf1Δ strain (DL3548) expressing PAF1, paf1-4A, or bearing empty vector. (G) Mpk1-HA ChIP on FKS2. The same strains as in (B), but expressing Mpk1-HA, were processed for ChIP analysis on FKS2 after heat stress.

Ki-Young Kim, et al. Cell. ;144(5):745-756.
7.
Figure 3

Figure 3. A Paf1 mutant that is defective for interaction with Mpk1. From: Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination.

(A) Yeast 2-hybrid interaction of Mpk1 with Paf1 fragments. An Mpk1-Gal4DBD fusion was tested for interaction with the indicated Paf1-Gal4AD fusion under conditions of cell wall stress. End-points of Paf1 fusions are indicated at top. Fusions that contained residues 85–131 (black) displayed β-galactosidase activities (U, units) higher than empty vector. (B) A Paf1 mutant that fails to bind Mpk1. A paf1Δ strain (DL3548), expressing the indicated forms of Mpk1 and Paf1, was treated as in Figure 2. (C) Mpk1 fails to associate with any Paf1C subunit in the paf1-4A mutant. A paf1Δ strain (DL3548) expressing Mpk1-Flag, the indicated TAP-tagged Paf1C subunit (TF-ZZ-HA) and either Paf1, or Paf1-4A, were processed for co-immunoprecipitation of Mpk1 with the various Paf1C subunits. (D) The paf1-4A mutant is specifically hyper-sensitive to cell wall stress. A paf1Δ strain (DL3792), expressing PAF1 (top of each panel), paf1-4A (middle), or bearing empty vector (bottom), was subjected to the indicated stresses on YPD plates, or unstressed (YPD). Stresses were: heat stress, HS (39°C); Congo Red (CR, 50 μg/ml); Calcofluor white (CFW, 20 μg/ml); thiabendazole (TBZ, 50 μg/ml); cycloheximide (CHX, 50ng/ml); LiCl (50mM); formamide (Form., 3%); caffeine (Caff., 8mM); MgCl2 (750mM); sodium orthovanadate (Van., 3mM); and hygromycin (Hyg., 20μg/ml). Equivalent cell numbers of each strain were spotted as serial 10-fold dilutions onto plates and incubated for three days at 30°C (or 39°C).

Ki-Young Kim, et al. Cell. ;144(5):745-756.

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