Functional analysis of Clp1 domains. (A) Schematic of mutations analyzed in this study. The linear representation of Pcf11 indicates the CID, the Q-rich domain (Q20), the regions which interact with Rna14, Rna15 and Clp1, the two regions containing zinc fingers (ZF), and the positions of the R480A and W482A mutations. The Clp1 representation shows the three domains determined by structural analysis (28), the N-terminal and C-terminal deletions, and the positions of mutations in the Pcf11 interaction interface and the ATP-binding site. (B) The amino and carboxyl termini of Clp1 are essential for cell viability. The CM246 [YCpURA3-CLP1] plasmid shuffle strain expressing epitope-tagged versions of wild-type Clp1 or the indicated deletion derivatives on a LEU2-marked plasmid were streaked on 5-FOA medium to counterselect the YCpURA3-CLP1 plasmid. Plates were photographed following incubation for 4 days at 24°C. (C) In vivo expression of truncated forms of Clp1. Extracts prepared from strains expressing either full-length Clp1 or the indicated deletion derivatives and grown on selective medium were analyzed by western blot using monoclonal antibody against the V5 tag on Clp1 or the CPF subunit Pta1 as a loading control. Asterisk indicates non-specific band detected by the V5 antibody. (D) Clp1 interacts with specific CPF subunits. ³⁵S-labeled in vitro-translated subunits of CPF were incubated with GST, GST-Clp1, GST-clp1(101–445) or GST-clp1(1–341) bound to glutathione–Sepharose beads. The bound proteins and 10% of the input were separated on a 10% polyacrylamide–SDS gel and detected by autoradiography.

Mutations in the Clp1 interaction interface of Pcf11 cause defects in mRNA 3′-end formation in vitro. (A) In vitro 3′-end processing assay. For coupled cleavage-polyadenylation assays (left side), extracts prepared from the strains containing the wild-type (PCF11) or the pcf11(RW-A) mutant grown at 30°C were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, unreacted precursor) for 20 min at 30°C. The same conditions were used for poly(A) addition assays (right side) except that the precleaved GAL7-9 RNA was used as precursor. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. Positions of substrate and product are indicated on the left and right. (B) The processing defect of pcf11(RW-A) is rescued by addition of CF IA. Coupled cleavage-polyadenylation assays were performed as described in Panel A using the indicated extracts. Recombinant CF IA containing wild-type Clp1, Pcf11, Rna14 and Rna15 was added to pcf11(RW-A) extract before initiating the reaction (right-most lane).

Analysis of mutations in the ATP-binding site of Clp1. (A) Mutations predicted to displace ATP cause inviability. The XH5 [YCpURA3-CLP1] plasmid shuffle strains expressing V5-tagged versions of wild-type Clp1 or the indicated mutants (K136E, T137W and K136E/T137W) were grown in selective medium, and 10-fold serial dilutions were spotted on 5-FOA plates and incubated for 4 days at 24°C. (B) In vivo expression of clp1 mutants. Extracts from strains expressing either wild-type Clp1 or the indicated mutants were analyzed by western blot using monoclonal antibody against the V5 tag or the CPF subunit Pta1 as a loading control. (C) Wild-type Clp1 and the clp1 (K136E,T137W) mutant proteins contain equivalent amounts of ATP. Equal amounts of wild-type and mutant recombinant proteins were TCA precipitated and the supernatant applied to a DNA Pac PA-100 (Dionex) HPLC column. Elution profiles were recorded at 260 nm for ATP alone and for ATP extracted from the acid-precipitated Clp1 mutant (red) and wild-type protein (green). ATP (blue) eluted at 0.57 M NaCl on a gradient of 0–1 M NaCl over 30 min. For clarity, the three traces were displaced with respect to one another on the Y-axis.

Regions and interactions of the yeast Clp1. (A) Summary of physical interactions known to occur between subunits of CF IA and other factors, indicated by connecting lines (Clp1 interactions, dashed line; Pcf11 interactions, solid black line; Rna14 interactions, dotted line; Rna15 interactions, gray line). These interactions include CPF subunits (19,51,52), and this study), Hrp1 (26), Pab1 (11), the CTD of the Rpb1 subunit of RNA Polymerase II (RNAP II) (20,21), and Yra1 of the mRNA export machinery (49). CPF subunits are indicated as rectangles; interactions of CF IA with CPF subunits in the right column have not been demonstrated. (B) Schematic diagram of Clp1 summarizing the regions needed for interaction with different CPF subunits. Pcf11 interacts with amino acids primarily in the central domain of Clp1 but partly in the CTD (28). (C) Model of the Clp1 ATP-binding site, based on the published structure (28). The locations of K136 and T137 are indicated. Parts of the surface composed of carbon are colored green, oxygen is red, and nitrogen is blue.

The pcf11 (RW-A) mutant is thermosensitive for growth and defective for Clp1 interaction. (A) Relative growth rates at different temperatures. Strains were grown in liquid YPD, and 10-fold serial dilutions were spotted on YPD plates and incubated for 2–3 days at the indicated temperatures. (B) In vivo expression of the pcf11 (RW-A) mutant. Cells containing a chromosomal deletion of the PCF11 gene but expressing V5-tagged forms of either Pcf11 or pcf11 (RW-A) or an untagged version of wild-type Pcf11 were grown at 30°C and then shifted to either 30°C or 39°C for an additional 60 min. Extracts from these cells were analyzed by western blot using a monoclonal antibody against the V5 tag or Pta1. (C) Two-hybrid analysis of the interaction between pcf11(RW-A) and Clp1. For two-hybrid analysis in yeast, plasmids carrying Pcf11, pcf11 (RW-A) fused to the Gal4 DNA-binding domain (GBD), or only the GBD (none) and plasmid carrying Clp1 fused to the Gal4 activation domain (pGAD-Clp1) were cotransformed into strain PJ69-4A. Ten-fold serial dilutions of the resulting strains were spotted on medium lacking uracil and leucine (CM-UL), as indicated. Activation of HIS3 expression was tested by spotting the same strains on medium lacking uracil, leucine and histidine (CM-ULH), and incubated for 2–3 days at 30°C. (D) Expression of GBD-Pcf11 and GAD-Clp1 analyzed by western blot. Extracts prepared from the different two-hybrid strains grown at 30°C were analyzed by western blot using a monoclonal antibody recognizing either the GAL4 activation or DNA-binding domain. (E) Pulldowns of Pcf11/Clp1 complexes in yeast extract. Extract was prepared from cells expressing V5-tagged forms of either Pcf11 or pcf11 (RW-A) and grown at 30°C. Immunoprecipitations from each extract were conducted using antibody against the V5 epitope, and blots of the precipitated proteins (IP) probed with Clp1 or Pcf11 antibody. The amount of Clp1 in the different extracts is shown in the panel marked ‘Input’.

Mutations in the Clp1 interaction interface of Pcf11 cause defects in mRNA 3′-end formation and transcription termination in vivo. (A) In vivo 3′-end processing and termination assay. Quantitative measurements of β-galactosidase activity were performed using wild-type or mutant strains transformed with reporter vectors lacking or containing an efficient polyadenylation signal upstream of the lacZ gene and grown at 30°C. The amount of read-through is expressed as a percentage of that obtained from vector lacking the poly(A) site. The data is the average of 48 independent assays for each strain. The numbers under the graph indicate the fold increase in read-through transcription over the wild-type value for each poly(A) site. (B) 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 1–9. (C) Chromatin immunoprecipitation analysis of Rpb3 crosslinking to PMA1. Cells are grown at 30°C and then shifted to either 30°C or 39°C for an additional 60 min. before formaldehyde crosslinking. Lane C denotes a non-transcribed 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 (41). The input signal represents DNA prior to immunoprecipitation. (D) Quantification of the data shown in panel B. Rpb3 occupancy of each of the indicated regions of PMA1 was calculated as a sample IP:input ratio, normalized to the IP:input ratio of the untranscribed region of chromosome V (lane C in RPB3 and INPUT panels) and plotted as fold increase over background (ratio = 1.0).

Clp1 proteins with K136E and T137W replacements are defective for Pcf11 interaction. (A) Two-hybrid analysis of the interaction between Pcf11 and different forms of Clp1. Two-hybrid analysis was performed as described in Figure 2C. (B) Pulldowns of Pcf11–Clp1 complexes in yeast extract. Extract was prepared from cells expressing V5-tagged forms of either Clp1 or clp1 (K136E, T137W) in addition to the untagged wt Clp1 needed for growth of the clp1(K136E, T137W) strain. Cells were grown at 30°C. Immunoprecipitations from each extract were conducted using antibody against the V5 epitope, and blots of the precipitated proteins (IP) probed with Pcf11 or Clp1 antibody. The amount of Pcf11 in the different extracts is shown in the panel marked ‘Input’.